Rhg1 mediated resistance to soybean cyst nematode

ABSTRACT

Methods of increasing the resistance of plants, in particular soybeans, to nematodes, in particular soybean cyst nematodes, are provided herein. The methods include increasing the expression of Glyma18g02580, Glyma18g02590 and/or Glyma18g2610 in cells of a plant and in particular in root cells of a plant to increase the resistance of the plant and plant cells to nematodes. The methods include increasing the expression using constitutive promoters or by increasing the copy number of the polynucleotides. Constructs for expressing these polypeptides, transgenic cells, transgenic plants and methods of generating the same are also provided. Methods of screening plant cells for resistance or susceptibility to nematodes are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of priority of U.S.Provisional Patent Application No. 61/646,017, filed May 11, 2012 andU.S. Provisional Patent Application No. 61/676,854, filed Jul. 27, 2012,which are both incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbers06-CRHF-0-6055 and 10-CRHF-0-6055 awarded by USDA/NIFA. The governmenthas certain rights in the invention.

BACKGROUND

Soybean cyst nematode (SCN) is currently the most economically damagingdisease for United States soybean production in most years. Estimatessuggest that SCN accounts for over $700 million in reduced soybeanproduction in the United States annually. SCN also seriously impactssoybean production in other countries such as Brazil, Argentina andChina. Soybean varieties with increased resistance to SCN have beenidentified, but resistance is quantitative and efficacy varies dependingon nematode genotypes, hence use of the more resistant varieties stillcan result in soybean yield loss due to SCN.

The genetic basis for resistance to SCN has been partially defined, tothe level of genetic loci, and appropriate sources of the soybean locusRhg1 make substantial contributions to SCN resistance. Prior to thepresent work, the specific genes and gene products controllingRhg1-mediated SCN resistance have not been successfully documented.

SUMMARY

Methods of increasing resistance of a plant to nematodes, in particularincreasing resistance of soybeans to SCN are provided herein. Severalgene products from the rhg1-b locus are identified and the relationshipof the gene products to resistance to SCN in soybeans is demonstrated.

In one aspect, methods of increasing resistance of a plant to nematodes,suitably cyst-forming nematodes, suitably SCN by increasing theexpression of or altering the expression pattern or gene copy number ofa polynucleotide encoding a Glyma18g02580 polypeptide, a Glyma18g02590polypeptide, a Glyma18g02610 polypeptide, a polypeptide having 90% ormore identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6,or SEQ ID NO: 3, or a homolog or functional variant of any of theaforementioned polypeptides in cells of the plant are provided. Use ofcombinations of the polypeptides is envisioned. The polynucleotidesencoding these polypeptide sequences may be derived from the Williams82, PI88788 or Peking (PI 548402) soybean varieties or other sources ofthe polynucleotides. The poly peptide sequences are provided and thepolymorphisms between the sequences in different varieties are noted.Increased expression of the polynucleotides in cells of the plantincreases the resistance of the plant to nematodes. Suitably expressionis increased in cells of the root of the plant. Suitably expression ofat least two of the polynucleotides is increased. Suitably, expressionof all three of the polynucleotides is increased.

In another aspect, methods of increasing resistance of a plant tonematodes, suitably cyst-forming nematodes, suitably SCN by altering(increasing or decreasing) the expression in cells in the root of theplant of a polypeptide identical or similar to at least a portion of SEQID NO: 1 of Glyma18g02580, SEQ ID NO: 2, 5 or 6 of Glyma18g02590 or SEQID NO: 3 of Glyma18g02610 relative to the expression in cells in theroot of the plant of a polypeptide whose expression can be used as acontrol, such as Glyma11g35820, are provided. Suitably expression of atleast two of the polypeptides is increased. Suitably, expression of allthree of the polypeptides is increased. Alternatively or in addition,expression of the polynucleotides encoding the polypeptides ofGlyma18g02610, Glyma18g02590, and/or Glyma18g2580 may be increased aswell.

In another aspect, methods of identifying plants that exhibit usefullevels of resistance of a plant to nematodes suitably cyst-formingnematodes, suitably SCN by identifying plants that exhibit altered(increased or decreased) expression in cells in the root of the plant ofa polypeptide identical or similar to at least a portion of SEQ ID NO: 1of Glyma18g02580, SEQ ID NO: 2, 5 or 6 of Glyma18g02590 or SEQ ID NO: 3of Glyma18g02610 relative to the expression in cells in the root of theplant of a polypeptide whose expression can be used as a control, suchas Glyma11g35820, are provided. Suitably expression of at least two ofthe polypeptides is at a higher level than in plants that are moresusceptible to SCN. Suitably, expression of all three of thepolypeptides is at a higher level. Alternatively or in addition,expression of the polynucleotides encoding the polypeptidesGlyma18g02610, Glyma18g02590, and/or Glyma18g2580 may be at a higherlevel as well.

In yet another aspect, a construct comprising a promoter operably linkedto a polynucleotide encoding at least a portion of Glyma18g02580polypeptide comprising SEQ ID NO: 1, a Glyma18g02590 polypeptidecomprising SEQ ID NO: 2, 5 or 6, a Glyma18g02610 polypeptide comprisingSEQ ID NO: 3 or a polypeptide having at least 90% identity to SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 or a homologor functional portion of any of the aforementioned polypeptides orcombinations thereof is provided. The construct may be used to generatetransgenic plants or seeds.

In still another aspect, a transgenic plant comprising an exogenous ornon-native polynucleotide encoding at least a portion of Glyma18g02580polypeptide comprising SEQ ID NO: 1, Glyma18g02590 polypeptidecomprising SEQ ID NO: 2, 5 or 6, Glyma18g02600 polypeptide comprisingSEQ ID NO: 4, Glyma18g02610 polypeptide comprising SEQ ID NO: 3 or apolypeptide having at least 90% identity to SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, a homolog or afunctional portion of any of the aforementioned polypeptides orcombinations thereof or the polypeptides described herein from eitherthe PI88788 or Peking-source is provided. The transgenic plant hasincreased resistance to nematodes, suitably cyst-forming nematodes,suitably SCN. Suitably, the transgenic plant comprises at least onepolynucleotide encoding at least two or at least three of thepolynucleotides encoding the Glyma18g02580, Glyma18g02590, andGlyma18g02610 polypeptides.

In a further aspect, a transgenic cell comprising a polynucleotideencoding a polypeptide capable of increasing resistance to nematodes,suitably cyst-forming nematodes, suitably SCN is provided. Thepolypeptide includes at least a portion of a polypeptide having at least90% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or similarsequences derived from PI88788 (such as SEQ ID NO: 5) or Peking-source(such as SEQ ID NO: 6) or combinations thereof. Suitably, thepolynucleotide includes at least two or three of the polypeptides havingat least 90% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.

In another aspect, methods of generating a transgenic plant byintroducing an exogenous polynucleotide encoding at least a portion of aGlyma18g02580 polypeptide having at least 90% identity to SEQ ID NO: 1,Glyma18g02590 polypeptide having at least 90% identity to SEQ ID NO: 2,5 or 6, or Glyma18g02610 polypeptide having at least 90% identity to SEQID NO: 3, or homologs or combinations thereof are provided. Thetransgenic plant has increased expression of Glyma18g02610,Glyma18g02590, and/or Glyma18g02580 in a cell in a root of the plant.The transgenic plant has increased resistance to nematodes, suitablycyst-forming nematodes, suitably SCN, as compared to a control plant.Suitably, the transgenic plant has increased expression of at least twoof the polynucleotides or all three of the polynucleotides encoding theGlyma18g02610, Glyma18g02590, and/or Glyma18g02580 polypeptides.

In yet a further aspect, methods of identifying molecules that interactwith the Rhg1 locus, Glyma18g02610, Glyma18g02590 and/or Glyma18g02580RNA transcripts, or the Glyma18g02610, Glyma18g02590 and/orGlyma18g02580 polypeptide are provided. The methods include detectingmolecules capable of binding the Rhg1 locus, Glyma18g02610,Glyma18g02590 or Glyma18g02580 RNA transcripts, or Glyma18g02610,Glyma18g02590 or Glyma18g02580 polypeptides.

in a still further aspect, methods of identifying the resistance orsusceptibility phenotype of a plant to cyst nematodes are provided. Themethod includes detecting a genetic marker associated with cyst nematoderesistance or susceptibility in a first plant cell and comparing thegenetic marker in the first plant cell to the genetic marker in a secondplant cell with a known resistance or susceptibility phenotype or acontrol plant cell. The genetic marker may be sequence variations,methylation differences, mRNA expression differences or otherdifferences identified herein. Suitably, the genetic marker isassociated with characteristics of the Rhg-1 locus, such as thosereported herein. Suitably, the genetic marker is the genomic copy numberof at least one of Glyma18g02600, Glyma18g02610, Glyma18g02590 orGlyma18g02580. Suitably the plant is a soybean and the nematodes areSCN.

In still a further aspect, methods of increasing resistance of a plantto nematodes comprising expressing a polynucleotide encoding aGlyma18g02610 polypeptide, a Glyma18g02590 polypeptide, or aGlyma18g02580 polypeptide, a polypeptide having 90% or more identity toSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 3,or a homolog or functional variant or combinations of any of theaforementioned polypeptides in a cell. Suitably, the polynucleotideencodes at least two or all three of the Glyma18g02610, Glyma18g02590 orGlyma18g02580 polypeptides. The polypeptides or a cell encoding thepolypeptide may then be applied to the plant, seeds of the plant or tosoil in which the seeds may be planted. The application increases theresistance of the plant to nematodes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pictorial depiction of the lifecycle of SCN.

FIG. 2 is a pictorial depiction of one gene silencing strategy that usesartificial microRNA sequences to target a gene of interest.

FIG. 3 shows a there are three genes at rhg1-b that contribute to SCNresistance.

FIG. 3A is a photograph showing representative SCN-infested roots; rootvascular cylinder and nematodes stained with acid fuchsin. Fewernematodes progress from J2 to J3, J4, adult male or egg-filled adultfemale (cyst) stages in SCN-resistant roots. FIG. 3B is a graph showingthat SCN development beyond J2 stage in transgenic roots of soybeanvariety Fayette with the designated gene silenced, relative to Williams82 (SCN-susceptible) and non-silenced Fayette (SCN-resistant) controls.Mean±std. error of mean. *: Fayette (silenced) significantly differentfrom Fayette (not silenced) based on ANOVA p<0.05. EV: transformed withempty vector.

FIG. 4 is a set of graphs showing that nematode development is impactedby level of silencing. FIG. 4A and 4C show that nematode development onWilliams 82 and Fayette roots transformed with empty vector (EV), orFayette transformed with silencing constructs (2580RNAi or ami2590) wasdependent on level of silencing. Transgenic roots with reduced targettranscript abundance (+) displayed nematode development similar toWilliams 82 (SCN-susceptible), while transgenic roots with non-silencedtranscript level (−) had nematode development similar to Fayette(SCN-resistant). FIGS. 4B and 4D show the transcript abundance of targetgenes in roots from (A) or (C) respectively, measured by qPCR. SKP16transcript used as reference and normalized to Fayette-EV. The resultsof FIGS. 4B and 4D were used to place roots in the ‘well-silenced’ (+)or ‘not well-silenced’ (−) categories shown in FIGS. 4A and 4C. FIGS. 4Aand 4B are Glyma18g02580, FIGS. 4C and 4D are Glyma18g02610. Barsrepresent mean±std. error of mean.

FIG. 5 shows a 31.2 kb repeat that elevates expression of the encodedgenes is present in SCN-resistant haplotypes of the Rhg1 locus. FIG. 5Ais a schematic of Rhg1 locus of Williams 82 (top), and five fosmidinserts from rhg1-b haplotype. DNA sequences of soybean reference genomeshown for the two designated locations. Numbers and block icons refer tosoybean genes (e.g., Glyma18g02540). Fosmids #3, 4 and 5 carry rhg1-bgenome segments that span repeat junctions. FIG. 5B shows the Rhg1repeat junction sequence from four different sources of SCN resistance(compare to reference genome sequences in (FIG. 5A)), FIG. 5C is a graphshowing the number of whole-genome shotgun sequencing readscorresponding to reference genome region shown in green in FIG. 5A wasten-fold greater than for genome regions adjacent to rhg1-b onchromosome 18 or for Rhg1-homeologous loci on chromosomes 11 and 2. FIG.5D is a graph showing transcript abundance of genes encoded in the 31 kbrepeat region is much greater in roots from SCN-resistant soybeanvarieties relative to SCN-susceptible varieties. Mean±std. error of meanshown for qPCR; results for Glyma18g02600 were at limit of detection

FIG. 6 shows Fiber-FISH detection of Rhg1 copy number variation inwidely used soybean lines. FIG. 6A is a schematic showing the twoadjacent probes isolated from a single PI88788 (rhg1-b) genomic DNAfosmid clone whose insert spans a repeat junction, generating a 25.2 kbprobe (green label) and an adjacent 9.7 kb probe (red label). DNA forgreen-labeled and red-labeled fiber-FISH probes are shown under thecorresponding sequence regions of Williams 82. The 25.2 kb fragment fromrhg1-b haplotype used for green probe was a single continuous DNAfragment that spans a repeat junction. FIG. 6B shows a composite of fourFiber-FISH images (four DNA fibers) per genotype, and probe diagram.Alternating pattern of red and green hybridization on single genomic DNAfibers indicates ten and three direct repeat copies of the 31 kb blockat Rhg1 locus of SCN-resistant Fayette (rhg1-b derived from PI 88788)and Peking (PI 548402) respectively, and one copy per Rhg1 haplotypeSCN-susceptible Williams 82. White bars=10 μm, which correspond toapproximately 32 kb using a 3.21 kb/μm conversion rate.

FIG. 7 shows that multiple SCN-resistant varieties contain the DNAjunction indicative of a repeat within the Rhg1 locus, and exhibitelevated expression of genes fully encoded within the repeat. FIG. 7A isa schematic of PCR primers used in FIG. 7B (see also FIG. 5). FIG. 7B isa photograph of a gel showing the results of PCR using outward-directedoligonucleotide primers shown in FIG. 7A that match sequences at theouter edges of the 31 kb segment of Rhg1 locus that is repeated in somesoybean varieties. R indicates SCN-resistant and S indicatesSCN-susceptible soybean variety. For primers 81 and 82 see Table 4. FIG.7C shows the DNA. Sequence from 11 SCN-resistant varieties and revealsidentical sequence for the repeat junction indicating a shared origin.Red bar indicates repeat junction (see also FIG. 5). FIG. 7D is a graphshowing the transcript abundance for genes encoded at Rhg1 (normalizedto SKP16), revealing elevated expression of genes fully encoded withinthe repeats of Rhg1 from PI 88788 or Peking sources, relative toexpression of the same genes in SCN-susceptible varieties. Barsrepresent mean±std. error of mean. Glyma18g02600 is expressed below0.01% of SKP16 (CT>35 cycles). FIG. 7E is an RNA blot analysis forGlyma18g02570 using RNA collected from roots of whole plants of Fayetteand Forrest (SCN resistant) and Williams 82 (SCN susceptible). * denotesthe band corresponding to the expected transcript size of Glyma18g02570(1.2 kb). The band at 1.8 kb corresponds to non-specific ribosomalbinding. Cultivars Fayette and Forrest (that contain repeats of the 31kb DNA segment) display the same banding pattern as Williams 82 (thatcontains a single copy of the 31 kb DNA segment); no alternativetranscripts for Glyma18g02570 were detected as a result of the repeatedDNA in Fayette and Forrest. RACE PCR from plants carrying rhg1-bconfirmed full-length transcripts (with transcript ends as annotated inthe reference genome) for Glyma18g02580, -2590 and -2610.

FIG. 8 is a graph showing qPCR for genes in and outside of Rhg1 repeat.RNA collected from roots of 3 individual plants grown in pots, 5 dayspost emrgence. Dark gray bars are estimated to be high copy number linesbased on gDNA qPCR and cDNA sequencing. Light grey bars are low copynumber containing lines that also require Rhg4 for full resistance.

FIG. 9 contains example gel photographs and a table summarizing manyexperiments showing that resistant and susceptible cultivars havedifferential DNA methylation at or adjacent to the genes in theduplicated region, especially in the promoter regions. In McrBCexperiments, methylated genomic DNA is cleaved by McrBC, which reducesthe abundance of the PCR product, while in HpaII experiments, methylatedgenomic DNA is not cleaved by HpaII and it is the non-methylated DNAthat is cleaved, leading to reduced abundance of the PCR product.

FIG. 10 is a photograph of a Western blot showing that an epitope-taggedversion of the Glyma18g02610 protein, produced from an introducedpolynucleotide in transgenic roots, is expressed in both Williams 82 andFayette transgenic roots and the products are similar in size.

FIG. 11A is a graph showing the quantitative PCR gene expressionanalysis for genes at the Rhg1 locus in susceptible and resistant rootsshowing that some of these genes not only are more highly expressed inresistant cultivars (as is also shown in FIG. 11), but also exhibit someupregulation after inoculation with SCN.

FIG. 11B is a graph showing the quantitative PCR gene expressionanalysis following methyl jasmonate or water treatment, which revealsthat Glyma18g02610 is expressed more highly in response to elevatedlevels of methyl jasmonate.

FIG. 12 is a set of photographs showing the histochemical staining ofpromoter-GUS expression in Fayette hairy root with (B, D, F and H) orwithout (A, C, E, and G) nematode inoculation. A and B showGlyma18g02580. C and D show Glyma18g02590. E and F show Glyma18g02610. Gand H show Glyma14g06080.

FIG. 13 provides the nucleotide and amino acid sequences forGlyma18g2590 from the indicated varieties.

FIG. 14 is a computer generated schematic of the three-dimensionalstructure of Glyma18g2590 showing the polymorphisms among the varietiesin the structure.

FIG. 15 is a graph showing elevated SCN resistance conferred bysimultaneous overexpression of multiple genes rather than overexpressionof individual genes from the 31 kb rhg1-b repeat. SCN development beyondJ2 stage is reported for transgenic soybean roots (variety Williams 82)overexpressing the designated single genes, or overexpressing all genesencoded within the 31 kb repeat (Glyma18g02580, -2590, -2600 and -2610),relative to Williams 82 (SCN-susceptible) and Fayette (SCN-resistant)controls. Mean±std, error of mean for roots transformed with emptyvector (EV) or gene overexpression constructs (OX). *: Williams 82-OXsignificantly different from Williams 82-EV based on ANOVA p<0.05.

FIG. 16 is a set of graphs showing that expressing the native FayetteGlyma18g02590 allele in Williams 82 does not alter SCN development. FIG.16A is a graph showing similar nematode development on transgenic rootsof Williams 82 expressing empty vector (EV) or Williams 82 expressingthe Fayette (rhg1-b-type) allele of Glyma18g02590 under control ofFayette Glyma18g02590 promoter sequences (2590_(FayP)::2590_(Fay)).Williams 82 transformed with either construct allowed a greaterproportion of nematodes to advance beyond the J2 stage compared toFayette-EV. FIG. 16B is a graph showing transcript abundance forGlyma18g02590 in roots from FIG. 16A, measured by qPCR. SKR16 transcriptused as reference; data normalized to Williams 82-EV. Bars in FIG. 16Aand FIG. 16B represent mean±std. error of mean.

FIG. 17 is a graph showing that qPCR reveals elevated transcriptabundance of the intended genes in roots transformed with the multiplegene simultaneous overexpression construct of FIG. 15, and nosignificant elevation of PR-1 expression. Transgenic roots carriedeither the multiple-gene construct (OX) or empty vector (EV). Similarresults obtained in second independent experiment with differenttransgenic events, except PR-1 abundance was more similar (closer to1.0) between Williams 82-EV, Fayette-EV and Williams-OX roots in secondexperiment. Bars represent mean±std. error of mean. Data forGlyma18g02600 are less dependable for Williams-EV and Fayette-EV becausetheir qPCR signal was at the limit of accurate qPCR detection (CT>33).

FIG. 18 is a set of graphs showing that overexpression of Glyma18g2580,Glyma18g2590 and Glyma18g2610 in combination can confer resistance on asusceptible Williams 82 variety.

DETAILED DESCRIPTION

Methods of identifying plants resistant or susceptible to cystnematodes, such as the soybean cyst nematode (SCN), methods of assessinga plant's level of resistance or susceptibility to SCN, methods ofincreasing resistance of a plant or plant cells to cyst nematodes andmethods of generating transgenic plant materials, including transgeniccells and plants, are provided herein. In addition, constructs includingpolynucleotides encoding the Rhg1 polypeptides described herein orhomologs or variants thereof are provided herein as SEQ ID NO: 1-6.Transgenic plants or transgenic plant cells with increased resistance tocyst nematodes, particularly SCN, carrying a transgene encoding anon-native or exogenous Rhg1 derived polynucleotide encoding thepolypeptides of SEQ ID NOs: 1-6 are provided herein. Non-transgenicplants carrying the polypeptides or bred or otherwise engineered toexpress increased levels of the polypeptides or the polynucleotidesencoding the polypeptides are also disclosed.

SCN is caused by the nematode Heterodera glycines. The life cycle of thenematode is shown in FIG. 1. Once afield is infested with this nematode,no economically feasible means of eliminating SCN from that fieldpresently exists. Current management of SCN often focuses on croprotation and planting SCN-resistant varieties of soybeans to control H.glycines populations across multiple years, as well as use ofSCN-resistant and/or SCN-tolerant soybeans to facilitate acceptableyield of the present year's crop. Practitioners have adopted “Race” and“Hg Type” terminologies to describe H. glycines populations according totheir ability to overcome known sources of plant SCN-resistance. Severalraces and Hg Types exist and soybean resistance to one type may offerlittle to no protection against another type of the nematode. Inaddition, H. glycines are outcrossing organisms for which localpopulations are genetically heterogeneous (and new nematode genotypescan be introduced), hence local populations can undergo shifts in raceor Hg Type such that previously effective plant SCN resistance can loseefficacy. Thus, the ability to identify which soybeans are resistant towhich H. glycines nematode populations and the further ability togenetically engineer soybean plants with increased resistance to morethan one type of nematode population is needed.

Soybeans with increased resistance to SCN are available and have beenused in cross-breeding experiments to generate soybeans that are moreresistant to SCN. The soybean rhg1 locus of Peking was previouslyidentified, mapped to a region of chromosome 18 (formerly known aslinkage group G), and a gene at that locus encoding a product carryingleucine-rich repeats and a protein kinase domain (LRR-kinase) washypothesized to account for the increased resistant to SCN. In plantscarrying the rhg1-b locus derived from soybean PI88788, SCN stillpenetrate and initiate feeding, but a high percentage of the syncitia donot persist and undergo the full sequence of nematode development(molting through the J3 and 14 stages to adulthood, sexualfertilization, and female transition to an embryo-filled andenvironmentally persistent cyst) (Li, Chen et al. 2004), (Colgrove andNiblack 2008). The molecular basis of this partial SCN-resistance is notunderstood. Despite 50 years of research on SCN, a pathogen causinghundreds of millions of dollars of economic losses in the U.S. annually,there were no confirmed public reports of a cloned soybean SCNresistance gene prior to the priority application.

Further fine genetic mapping of the rhg1 locus, which is also known asthe Rhg1 locus, or by other more restricted designations such as rhg1-b,was completed in plants carrying the PI88788 source of Rhg1, and newmarkers associated with the resistance genotype were identified. SeeKim, M., D. L. Hyten, A. F. Bent and B. W. Diers, 2010. Fine mapping ofthe SCN resistance locus rhg1-b from PI 88788. Plant Genome 3:81-89,which is incorporated herein by reference in its entirety. PI88788 waschosen because it is the source of resistance in many cross-bred linescurrently marketed as resistant to SCN. These markers are tightly linkedwith resistance or susceptibility to SCN and may be useful to identifyor predict whether soybean breeding lines are likely to display aSCN-resistant or SCN-sensitive phenotype. The refined map of the rhg1-blocus from PI88788 suggested that the LRR-kinase gene that is very closeto the rhg1-b locus does not make significant contributions to the SCNresistance phenotype. The study of Melito et al. 2010, which usedtransgenic roots expressing full-length transcripts or constructs thatpartially silence the expression of transcripts, also found no evidenceto support a role for the rhg1-b-proximal Glyma18g02680 LRR-kinase SCNresistance. Kim et al. demonstrated that the rhg1-b genetic componentsassociated with the SCN resistance/susceptibility phenotype of PI88788and its derivatives are located within the chromosomal interval definedby the termini BARCSOYSSR_(—)18_(—)0090 and BARCSOYSSR_(—)18_(—)0094.The most recent fine-structure genetic mapping defined an interval forrhg1-b that corresponds to a 67 kb interval carrying 11 predicted genesin the sequenced genome of SCN-susceptible Williams 82 soybean (Kim,Hyten et al. 2010). See FIG. 5.

Here we report the identification and functional testing of multiplegenes in the rhg1-b genetic interval. Within the Rhg1 locus, multiplecopies (ten, seven or three copies in the varieties investigated todate) of a chromosome segment encoding four identified genes within theRhg1 locus are present in SCN-resistant soybean varieties, while onlyone copy of this segment is present in the tested SCN-susceptiblevarieties that lack Rhg1 alleles derived from the resistant varietiessuch as PI88788, PI437654 or Peking. See FIGS. 5 and 6. Silencing of anyone of three genes within the multi-copy gene block using miRNA leads toincreased susceptibility to SCN in transgenic soybean roots. Intransgenic roots from a previously SCN-susceptible soybean variety,simultaneous overexpression of three or four of the rhg1-b genes fromthe multi-copy gene block leads to increased SCN resistance. The geneswithin this block are expressed at significantly higher levels in thetested SCN-resistant soybean varieties. Trans-acting factors in Fayettealso are not sufficient to drive the elevated expression of transgenicDNAs sequences carrying these ˜2 kb of Glyma18g02590 or Glyma18g02610promoter DNA sequence, when those sequences are integrated at loci otherthan rhg1-b in Fayette. DNA methylation at multiple sites within theRhg1 locus is polymorphic between SCN-resistant and SCN-susceptiblelines, and this may contribute to the gene expression differences thatcorrelate with SCN resistance. The number of copies of this locus alsocorrelates to the levels of expression of the Glyma18g2580, Glyma18g2590and Glyma18g2610 polypeptides and mRNAs and to the level of resistanceto SCN. Thus gene dosing based on increasing the number of copies of therepeated region of the DNA may be a key factor mediating increasedexpression of the polypeptides and increased resistance to SCN. Man yportions of these findings were reported in Cook, D. E., Lee, T. G.,Guo, X., Melito, S., Wang, K., Bayless, A., Wang, J., Hughes, T. J.,Willis, D. K., Clemente, T., Diers, B. W., Hudson, M. E. and Bent, A. F.2012. Copy Number Variation of Multiple Genes at Rhg1 Mediates NematodeResistance in Soybean. Science 338:1206-1209 and the associatedSupporting Online Material (Supplementary Materials) found atwww.sciencemag.org/content/suppl/2012/10/10/science.1228746.DC1.html,which are incorporated herein by reference in their entirety.

The resistance or susceptibility phenotype of a plant can be predictedwith valuable accuracy by comparing a genetic marker in the plant to thesame genetic marker or selectable marker in a second plant with knownresistance or susceptibility phenotype. Thus methods of screening afirst plant or plant cell for resistance or susceptibility to cystnematodes is provided herein. The methods include detecting a genetic orselectable marker associated with cyst nematode resistance orsusceptibility to cyst nematodes in the first plant cell and using thatmarker to predict the resistance or susceptibility of the first plant orplant cell to nematodes. Prediction does not mean a 100% guarantee ofthe phenotype regarding resistance or susceptibility of the plant tocyst nematodes. The predicting step may include comparing the marker inthe first plant or plant cell to the marker in a second plant or plantcell with a known resistance or susceptibility phenotype. The markerphenotype or genotype of the second cell is predictive of the cystnematode resistance phenotype in the first cell. The prediction may beused to select resistant soybeans or resistant plant cells for use ingenerating resistant soybean lines.

A plant includes any portion of the plant including but not limited to awhole, plant, a portion of a plant such as a part of a root, leaf, stem,seed, pod, flower, cell, tissue or plant germplasm or any progenythereof. Germplasm refers to genetic material from an individual orgroup of individuals or a clone derived from a line, cultivar, varietyor culture. Soybean plant refers to whole soybean plant or portionsthereof including, but not limited to, soybean plant cells, soybeanplant protoplasts, soybean plant tissue culture cells or calli. A plantcell refers to cells harvested or derived from any portion of the plantor plant tissue culture cells or calli.

The rhg1 locus is a chromosomal region identified as a region importantfor resistance to SCN. A locus is a chromosomal region where one or moretrait determinants, genes, polymorphic nucleic acids, or markers arelocated. A quantitative trait locus (QTL) refers to a polymorphicgenetic locus where the underlying gene controls a trait that isquantitatively measured and contains at least two alleles thatdifferentially affect expression of a phenotype or genotype in at leastone genetic background, with said locus accounting for part but not allof the observed variation in the overall phenotypic trait that is beingassessed. A genetic marker is a nucleotide sequence or amino acidsequence that may be used to identify a genetically linked locus, suchas a QTL. Examples of genetic markers include, but are not limited to,single nucleotide polymorphisms (SNP), simple sequence repeats (SSR; ormicrosatellite), a restriction enzyme recognition site change, genomiccopy number of specific genes or target sequences or other sequencebased differences between a susceptible and resistant plant.

Genetic or selectable markers can be detected using a variety ofanalytic methods, including RFLP, AFLP, sequence analysis, hybridizationsuch as allele specific hybridization analysis, differential PCR orother methods such as those known to those of skill in the art. A listof single nucleotide polymorphisms between resistant and susceptiblesoybeans in the Rhg1 multi-gene copy region is provided in Table 3. Inanother embodiment, the marker is the genomic copy number, or anestimate of the genomic copy number, of at least one of the genes or DNAsequences found in the replicated region of the resistant lines, in yetanother embodiment the marker is the genomic DNA segment carrying theborder between the replicated region at Glyma18g02610 and Glyma18g02570as shown in FIGS. 5 and 6. Selection methods may also include analysisof traits, phenotype polymorphisms selectable markers not defined by DNAor RNA sequence differences, such as differences in methylation of asequence, or polypeptide expression levels or in gene expression levels.As shown in the Examples the soybean SCN resistance Rhg1 locus, inparticular the promoter regions of Glyma18g02610, Glyma18g02590 andGlyma18g02580, was highly methylated in the resistant plants as comparedto susceptible plants. Methylation distinctions in and adjacent to thesegenes, for example in the promoter and upstream regions of the genes,may be used to distinguish between resistant and susceptible lines. Inaddition, resistant plants had higher mRNA levels for Glyma18g02610,Glyma18g02590 and Glyma18g02580 than susceptible plants. See FIG. 5.Thus methods of detecting the gene expression levels of any of thesegenes, for example by monitoring mRNA abundance, may be used in themethods described herein. In another embodiment, the marker may be theprotein expression level of at least one of Glyma18g02610, Glyma18g02590and Glyma18g02580. Any of these differences may be used as a screen totest whether a plant or plant cell is likely to he resistant orsusceptible to nematodes.

The markers described above are linked to the phenotype of increasedresistance to cyst nematodes or alternatively to susceptibility to cystnematodes. The methods of detecting may comprise amplifying the markeror a portion thereof to produce an amplified product. The presence ofthe product may be indicative of the marker or the amplified product maybe sequenced. The amplified product may also be assessed viadifferential sensitivity to a restriction endonuclease. The marker maybe detected using allele specific hybridization analysis, quantitativePCR, Northern blot analysis, Western blot analysis or anothermethodology. Methods of detecting or evaluating genetic or phenotypicmarkers of traits such as those descried herein are available to thoseof skill in the art, many such methods are provided in the Examples, andit is anticipated that new methods may be developed in the future todetect the Rhg1 polymorphisms described herein. For example, the markerscan be used to detect the presence or absence of the multi-copy Rhg1region during breeding selection processes.

A linked locus describes a situation in which a genetic marker and atrait are closely linked chromosomally such that the genetic marker andthe trait do not independently segregate and recombination between thegenetic marker and the trait does not occur during meiosis with a highfrequency. The genetic marker and the trait may segregate independently,but generally do not. For example, a genetic marker for a trait may onlysegregate independently from the trait 5% of the time; suitably only 5%,4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less of the time. Genetic markerswith closer linkage to the trait-producing locus will serve as bettermarkers because they segregate independently from the trait less oftenbecause the genetic marker is more closely linked to the trait. Geneticmarkers that directly detect polymorphic nucleotide sites that causevariation in the trait of interest are particularly useful for theiraccuracy in marker-assisted plant breeding. Thus, the methods ofscreening provided herein may be used in traditional breeding,recombinant biology or transgenic breeding programs or any hybridthereof to select or screen for resistant varieties.

In the methods described herein the SCN resistance or susceptibilityphenotype of a first soybean is identified by comparing the geneticmarker in the first soybean to that in a second soybean with a knownresistance phenotype. The second soybean may be known to be resistant toSCN. Thus a first soybean having the same genetic marker as the secondsoybean is likely to also be resistant to SCN. Resistant soybeans areknown in the art and include but are not limited to PI88788, Peking,Hartwig, Fayette, Forrest, LD02-5320, LD02-5025, and LD01-7323 or linescarrying loci that contributed to or were derived from these cultivarssuch as those provided in Table 2. In particular, the methods allowidentification of soybean plants having increased resistance to Race 3SCN and other nematode populations, similar to PI88788. Alternatively,the second soybean may be known to be susceptible to SCN. Thus a firstsoybean having the same genetic marker as the second soybean is likelyto be susceptible to SCN. Susceptible soybeans are known in the art andinclude, but are not limited to, ‘Williams 82’, Essex, Thorne, Sturdy,LG03-1672, and LG00-3372 or lines carrying loci that contributed to orwere derived from one of these cultivars such as those provided in Table2. In particular, the methods allow identification of soybean plantshaving susceptibility to SCN similar to that of ‘Williams 82.’ Althoughresistance to SCN is widely observed to be a quantitative trait, theterms susceptibility and resistance as used in the preceding paragraphsrefer to qualitative trans, such that identification as a resistantsoybean indicates that the soybean is more resistant than thesusceptible soybean line to which it is being compared. Likewise,identification of a soybean as a susceptible soybean indicates that thesoybean is more sensitive than the resistant soybean line to which it isbeing compared.

Resistance (or susceptibility) to SCN can be measured in a variety ofways, several of which are known to those of skill in the art. In theexamples, soybean roots were experimentally inoculated with SCN and theability of the nematodes to mature (molt and proceed to developmentalstages beyond the J2) on the roots was evaluated as compared to asusceptible and/or resistant control plant. A SCN greenhouse test isalso described in the Examples and provides an indication of the numberof cysts on a plant and is reported as the female index. Increasedresistance to nematodes can also be manifested as a shift in theefficacy of resistance with respect to particular nematode populationsor genotypes. Additionally but not exclusively, SCN-susceptible soybeansgrown on SCN-infested fields will have significantly decreased cropyield as compared to a comparable SCN-resistant soybean. Improvement ofany of these metrics has utility even if all of the above metrics arenot altered.

As demonstrated in the Examples a set of three genes found on a tandemlyrepeated segment of chromosome 18 were identified whose silencing led toincreased susceptibility to SCN in a resistant variety. The three genesare found along with a fourth gene, part of a fifth gene, and other DNAsequences in a chromosome segment approximately 31 kb in length that ispresent in 10 copies in the soybean varieties that carry the rhg1-ballele or haplotype of Rhg1 that is in widespread commercial use forcontrol of SCN disease of soybean. This Rhg1 chromosome segment is foundin at least three copies in all SCN resistant varieties tested to date.Various resistant varieties early three, seven or ten copies and thehigher copy number versions of Rhg1 express higher levels of transcriptsfor the three genes. Higher copy number versions of Rhg1 also confermore resistance to SCN on their own (exhibit less reliance on thesimultaneous presence of desirable alleles of other SCN resistance QTLsuch as Rhg4 in order to effective confer SCN resistance, relative toRhg1 haplotypes with lower Rhg1 repeat copy numbers). In the Examples,over-expression of the three genes in a susceptible variety made rootsmore resistant to SCN. Methods of increasing resistance of a plant tocyst nematodes by selecting plants carrying genetic markers associatedwith Glyma18g02610, Glyma18g02590, and/or Glyma18g02580 alleles that arepresent within the Rhg1 locus are described. As shown in the Examples,genetic polymorphisms ranging from single nucleotide polymorphisms togene rearrangements (i.e. gene duplications) and differences inmethylation may occur in other Glycine max plant lines and other Glycinespecies, which may alter the expression or biological impact of one ormore genes linked to the Rhg1 locus, and careful selection of desirablealleles of particular genes at the Rhg1 locus may be desirable to allowselection of plants with increased resistance to SCN.

Methods of increasing resistance of a plant to cyst nematodes, includingbut not limited to SCN, by increasing the expression of or altering theexpression pattern of or increasing the copy number of a polynucleotideencoding the Glyma18g02610 (SEQ ID NO: 3), Glyma18g02590 (SEQ ID NOs: 2,5 and 6), and/or Glyma18g02580 (SEQ ID NO: 1) polypeptides or functionalfragments or variants thereof in cells of the plant are also provided.The polypeptide may be 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the sequences provided. We have sequenced these genes fromboth resistant and susceptible varieties and found few polymorphismswithin the coding regions and few changes that result in an amino acidchange. The Glyma18g2590 polypeptide does have some significantpolymorphisms between the resistant and susceptible varieties thatappear to be functionally related to SCN resistance as shown in theExamples.

Suitably the expression of the polypeptides encoded by Glyma18g02610,Glyma18g02590, and/or Glyma18g02580 is increased in a root of the plant.Suitably, the expression of the polypeptides encoded by Glyma18g02610,Glyma18g02590, and/or Glyma18g02580 is increased in root cells of theplant. The plant is suitably a soybean plant or portions thereof. Thepolynucleotides may also be transferred into other non-soybean plants,or homologs of these polypeptides or polynucleotides encoding thepolypeptides from other plants, or synthetic genes encoding productssimilar to the polypeptides encoded by Glyma18g02610, Glyma18g02590,and/or Glyma18g02580 may be overexpressed in those plants. Other plantsinclude but are not limited to sugar beets, potatoes, corn, peas, andbeans. The overexpression of the genes may increase the resistance ofplants from these other species to nematodes and in particular cystnematodes, such as the soybean cyst nematode Heterodera glycines, thesugar beet cyst nematode Heterodera schacthii, the potato cyst nematodesGlobodera pallida and related nematodes that cause similar disease onpotato such as Globodera rostochiensis, the corn cyst nematodeHeterodera zeae, and the pea cyst nematode Heterodera goettingiana.

The expression of the polynucleotides may be increased by increasing thecopy number of the polynucleotide in the plant, in cells of the plant,suitably root cells, or by identifying plants in which this has alreadyoccurred. Suitably, the polynucleotide is present in three, seven oreven ten copies. Suitably at least two or all three of thepolynucleotides encoding the polypeptides or the polypeptides ofGlyma18g02610, Glyma18g02590, and Glyma18g02580 are expressed.Alternatively the expression may be increased using recombinant DNAtechnology, e.g., by using a strong promoters to drive increasedexpression of one or more polynucleotides.

In addition, methods of increasing resistance of a plant to cystnematodes may be achieved by cloning sequences upstream fromGlyma18g02610, Glyma18g02590, and/or Glyma18g02580 from resistant linesinto susceptible lines. For these methods, nucleotide sequences havingat least 60%, 70% or 80% identity to nucleotide sequences that flank theprotein-coding regions of Glyma18g02610, Glyma18g02590 or Glyma18g02580(or sequences having at least 80%, 85%, or 90% identity to thoseprotein-coding regions), said flanking regions including 5′ and 3′untranslated regions of the mRNA for these genes, and also including anyother genomic DNA sequences that extend from the protein coding regionof these genes to the protein coding regions of immediately adjacentgenes may be used.

The increase in expression of Glyma18g02610, Glyma18g02590, and/orGlyma18g02580 in the plant may be measured at the level of expression ofthe mRNA or at the level of expression of the polypeptide encoded byGlyma18g02610, Glyma18g02590, and/or Glyma18g02580. The level ofexpression may be increased relative to the level of expression in acontrol plant as shown in the Examples. The control plant may be anSCN-susceptible plant or an SCN-resistant plant. For example, asusceptible plant such as ‘Williams 82’ may be transformed with anexpression vector such that the roots of the transformed plants expressincreased levels of Glyma18g02610, Glyma18g02590, and/or Glyma18g02580as compared to an untransformed plant or a plant transformed with aconstruct that does not change expression of Glyma18g02610,Glyma18g02590, and/or Glyma18g02580, resulting in increased resistanceto nematodes. Alternatively, the control may be a plant partiallyresistant to nematodes and increased expression of Glyma18g02610,Glyma18g02590, and/or Glyma18g02580 may result in increased resistanceto nematodes. Alternatively, the plant may be resistant to nematodes andincreasing expression of Glyma18g02610, Glyma18g02590, and/orGlyma18g02580 may result in further increased resistance to nematodes.Alternatively, the plant may be more resistant to certain nematodepopulations, races, Hg types or strains and less resistant to othernematode populations, races, Hg types or strains, and increasingexpression of Glyma18g02610, Glyma18g02590, and/or Glyma18g02580 mayresult in increased resistance to certain of these nematode populations,races, Hg types or strains.

In the Examples, a decrease in expression of Glyma18g023610,Glyma18g02590, and/or Glyma18g02580 is shown to increase thesusceptibility of a SCN-resistant soybean to SCN maturation. Inaddition, roots of the susceptible ‘Williams 82’ soybean are shown tohave lower levels of Glyma18g02610, Glyma18g02590, and/or Glyma18g02580mRNA as compared to the resistant Fayette line. Because low levels ofGlyma18g02610, Glyma18g02590, and/or Glyma18g02580 mRNA correlate withnematode susceptibility, and increased levels correlate with resistance,and direct towering of Glyma18g02610, Glyma18g02590, and/orGlyma18g02580 mRNA is causally associated with greater nematodesusceptibility of previously resistant tissues, increasing the levels ofGlyma18g02610, Glyma18g02590, and/or Glyma18g02580 in a soybean shouldin many instances increase the resistance of the soybean to nematodes,in particular SCN. In FIG. 15, increased expression of a combination ofGlyma18g02600, Glyma18g02610, Glyma18g02590, and Glyma18g02580 was shownto increase resistance to SCN of a susceptible line. Increasedexpression of three genes, Glyma18g02610. Glyma18g02590, andGlyma18g02580 was also shown to increase resistance of an SCNsusceptible variety in FIG. 18. Increased expression of fewer than thesethree polynucleotides or of the polypeptides encoded by thepolynucleotides may be similarly effective to increase resistance.

Expression of Glyma18g02610, Glyma18g02590, and/or Glyma18g02580 may beincreased in a variety of ways including several apparent to those ofskill in the art and may include transgenic, non-transgenic andtraditional breeding methodologies. For example, the expression of thepolypeptide encoded by Glyma18g02610, Glyma18g02590, and/orGlyma18g02580 may be increased by introducing a construct including apromoter operational in the plant operably linked to a polynucleotideencoding the polypeptide into cells of the plant. Suitably, the cellsare root cells. Alternatively, the expression of the polypeptide encodedby Glyma18g02610, Glyma18g02590, and/or Glyma18g02580 may be increasedby introducing a transgene including a promoter operational in the plantoperably linked to a polynucleotide encoding the polypeptide into cellsof the plant. The promoter may be a constitutive or inducible promotercapable of inducing expression of a polynucleotide in all or part of theplant, plant roots or plant root cells. In another embodiment, theexpression of Glyma18g0208 Glyma18g02590, and/or Glyma18g02580 may beincreased by increasing expression of the native polypeptide in a plantor in cells of the plant, such as the plant root cells. In anotherembodiment, the expression of Glyma18g02610, Glyma18g02590, and/orGlyma18g02580 may be increased by increasing expression of the nativepolypeptide in a plant or in cells of the plant such as the nematodefeeding site, the syncitium, or cells adjacent to the syncitium. Inanother embodiment, the expression of Glyma18g02610, Glyma18g02590,and/or Glyma18g02580 may be increased by increasing expression of thenative polypeptide in a plant or in cells of the plant such as sites ofnematode contact with plant cells. In another embodiment, expression maybe increased by increasing the copy number of Glyma18g02610,Glyma18g02590, and/or Glyma18g02580. Other mechanisms for increasing theexpression of Glyma18g02610, Glyma18g02590, and/or Glyma18g02580include, but are not limited to, increasing expression of atranscriptional activator, reducing expression of a transcriptionalrepressor, addition of an enhancer region capable of increasingexpression of Glyma18g02610, Glyma18g02590, and/or Glyma18g02580,increasing mRNA stability, altering DNA methylation, histone acetylationor other epigenetic or chromatin modifications in the vicinity of therelevant genes, or increasing protein or polypeptide stability.

In addition to the traditional use of transgenic technology to introduceadditional copies or increase expression of the genes and mediate theincreased expression of the polypeptides of Glyma18g02610,Glyma18g02590, and/or Glyma18g02580 in plants, transgenic ornon-transgenic technology may be used in other ways to increaseexpression of the polypeptides. For example, plant tissue culture andregeneration, mutations or altered expression of plant genes other thanGlyma18g02610, Glyma18g02590, and/or Glyma18g02580, or transgenictechnologies, can be used to create instability in the Rhg1 locus or theplant genome more generally that create changes in Rhg1 locus copynumber or gene expression behavior. The new copy number or geneexpression behavior can then be stabilized by removal of thevariation-inducing mutations or treatments, for example by further plantpropagation or a conventional cross. In one of the examples, although atransgenic plant was used to create the change in copy number, theresult would be anon-transgenic line (and conceivably regulated as suchwith enhanced resistance due to increased copy number of the locus.Examples of transgenic technologies that might be used in this wayinclude targeted zinc fingers, ribozymes or other sequence-targetedenzymes that create double stranded DNA breaks at or close to the Rhg1locus, the cre/loxP system from bacteriophage lambda, TranscriptionActivator-Like Effector Nucleases (TALENs), artificial DNA or RNAsequences designed to recombine with Rhg1 that can be introducedtransiently, or enzymes that “shuffle” DNA such as the mammalian Rag1enzyme or DNA transposases. Mutations or altered expression ofendogenous plant genes involved in DNA recombination, DNA rearrangementand/or DNA repair pathways are additional examples.

The screening methods described above could also be used to screensoybean isolates (Glycine max) and closely related species (Glycinesoja, Glycine tomentella or other Glycine species) for resistancemarkers and then resistant lines can be crossed naturally orartificially with soybean to develop a soybean with a variant copynumber or sequence at the Rhg1 site. Any useful alleles identified insuch screens could then be introduced using traditional breeding ortransgenic technology into soybeans.

Non-transgenic means of generating soybean varieties carrying traits ofinterest such as increased resistance to SCN are available to those ofskill in the art and include traditional breeding, chemical or othermeans of generating chromosome abnormalities, such as chemically inducedchromosome doubling and artificial rescue of polyploids followed bychromosome loss, knocking-out DNA repair mechanisms or increasing thelikelihood of recombination or gene duplication by generation ofchromosomal breaks. Other means of non-transgenetically increasing theexpression or copy number of Glyma18g02610, Glyma18g02590, and/orGlyma18g02580 include the following: screening for mutations in plantDNA encoding miRNAs or other small RNAs, plant transcription factors, orother genetic elements that impact Glyma18g02610, Glyma18g02590, and/orGlyma18g02580 expression; screening large field or breeding populationsfor spontaneous variation in copy number or sequence at Rhg1 byscreening of plants for nematode resistance, Rhg1 copy number or otherRhg1 gene or protein expression traits as described in precedingparagraphs; crossing of lines that contain different or the same copynumber at Rhg1 but have distinct polymorphisms on either side, followedby selection of recombinants at Rhg1 using molecular markers from twodistinct genotypes flanking the Rhg1 locus; chemical or radiationmutagenesis or plant tissue culture/regeneration that creates chromosomeinstability or gene expression changes, followed by screening of plantsfor nematode resistance, Rhg1 copy number or other Rhg1 gene or proteinexpression traits as described in preceding paragraphs; or introductionby conventional genetic crossing of non-transgenic loci that create orincrease genome instability into Rhg1-containing lines, followed byscreening of plants for either nematode resistance or Rhg1 copy number.Examples of loci that could be used to create genomic instabilityinclude active transposons (natural or artificially introduced fromother species), loci that activate endogenous transposons (for examplemutations affecting DNA methylation or small RNA processing such asequivalent mutations to met1 in Arabidopsis or mop1 in maize), mutationof plant genes that impact DNA repair or suppress illegitimaterecombination such as those orthologous or similar in function to theSgs1 helicase of yeast or RecQ of E. coli, or overexpression of genessuch as RAD50 or RAD52 of yeast that mediate illegitimate recombination.Those of skill in the art may find other transgenic and non-transgenicmethods of increasing expression of Glyma18g02610, Glyma18g02590, and/orGlyma18g02580.

The polynucleotides and/or polypeptides described and used herein mayencode the full-length or a functional fragment of Glyma18g02610,Glyma18g02590, and/or Glyma18g02580 from the rhg1-b locus, or anaturally occurring or engineered variant of Glyma18g02610,Glyma18g02590, and/or Glyma18g02580, or a derived polynucleotide orpolypeptide all or part of which is based upon nucleotide or amino acidcombinations similar to all or portions of Glyma18g02610, Glyma18g02590,and/or Glyma18g02580 or their encoded products. Additionalpolynucleotides encoding polypeptides may also be included in theconstruct such as Glyma18g02600 (which encodes the polypeptide of SEQ IDNO: 4). The polypeptide may be at least 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% identical to the sequences provided herein. Thepolynucleotides encoding the polypeptides may be at least 50%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the sequencesavailable in the public soybean genetic sequence database.

The expression of the polypeptide encoded by Glyma18g02610,Glyma18g02590, and/or Glyma18g02580 may be increased, suitably the levelof polypeptide is increased at least 1.2, 1.5, 1.7, 2, 3, 4, 5, 7, 10,15, 20 or 25 fold in comparison to the untreated, susceptible or othercontrol plants or plant cells. Control cells or control plants arecomparable plants or cells in which Glyma18g02610, Glyma18g02590, and/orGlyma18g02580 expression has not been increased, such as a plant of thesame genotype transfected with empty vector or transgenic for a distinctpolynucleotide.

Increased resistance to nematodes may be measured as described above.The increased resistance may be measured by the plant having a lowerpercentage of invading nematodes that develop past the J2 stage, a lowerrate of cyst formation on the roots, reduced SCN egg production withincysts, reduced overall SCN egg production per plant, and/or greateryield of soybeans on a per-plant basis or a per-growing-area basis ascompared to a control plant grown in a similar growth environment. Othermethods of measuring SCN resistance also will be known to those withskill in the art. In the methods of increasing resistance to nematodesdescribed herein, the resulting plant may have at least 10% increasedresistance as compared to the untreated or control plant or plant cells.Suitably the increase in resistance is at least 15%, 20%, 30%, 50%,100%, 200%, 500% as compared to a control. Suitably, the female index ofthe plant with increased resistance to nematodes is about 80% or less ofthe female index of an untreated or control plantplant derived from thesame or a similar plant genotype, infested with a similar nematodepopulation within the same experiment. More suitably, the female indexafter experimental infection is no more than 60%, 40%, or 20% of that ofthe control plant derived from the same or a similar plant genotype,infested with a similar nematode population within the same experiment.Suitably, when grown in fields heavily infested with SCN (for example,more than 2500 SCN eggs per 100 cubic centimeters of soil), soybeangrain yields of field-grown plants are 2% greater than isogenic controlplants. More suitably, the grain yield increase is at least 3%, 4%, or5% over that of isogenic control plants grown in similar environments.

Also provided herein are constructs including a promoter operably linkedto a Glyma18g02610, Glyma18g02590, and/or Glyma18g02580 polynucleotideencoding a polypeptide comprising SEQ ID NO: 1-3 or 5-6 or a fragment orfunctional variant thereof. Also included are homologs or variants ofthese sequences from other soybean varieties. The constructs may furtherinclude Glyma18g02600 or other genes. The constructs may be introducedinto plants to make transgenic plants or may be introduced into plants,or portions of plants, such as plant tissue, plant calli, plant roots orplant cells. Suitably the promoter is a plant promoter, suitably thepromoter is operational in root cells of the plant. The promoter may betissue specific, inducible, constitutive, or developmentally regulated.The constructs may be an expression vector. Constructs may be used togenerate transgenic plants or transgenic cells. The polypeptide may beat least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to thesequences of SEQ ID NO: 1-3 or 5-6. The constructs may comprise allthree polynucleotides and may mediate expression of all threepolypeptides.

Transgenic plants including a non-native or exogenous polynucleotideencoding the rhg1-b polypeptides identified and described herein arealso provided. Suitably the transgenic plants are soybeans. Thetransgenic plants express increased levels of Glyma18g02610,Glyma18g02590, and/or Glyma18g02580 polypeptide as compared to a controlnon-transgenic plant from the same line, variety or cultivar or atransgenic control expressing a polypeptide other than Glyma18g02610,Glyma18g02590, and/or Glyma18g02580. The transgenic plants also haveincreased resistance to nematodes, in particular SCN, as compared to acontrol plant. Portions or parts of these transgenic plants are alsouseful. Portions and parts of plants includes, but is not limited to,plant cells, plant tissue, plant progeny, plant asexual propagates,plant seeds.

Transgenic plant cells comprising a polynucleotide encoding apolypeptide capable of increasing resistance to nematodes such as SCNare also provided. Suitably the plant cells are soybean plant cells.Suitably the cells are capable of regenerating a plant. The polypeptidecomprises the sequences of SEQ ID NOs: 1-3 or 5-6 or fragments, variantsor combinations thereof. The polypeptide may be 85%, 90%, 95%, 97%, 98%,99% or 100% identical to the sequences provided. The transgenic cellsmay be found in a seed. A plant, such as a soybean plant, may includethe transgenic cells. The plant may be grown from a seed comprisingtransgenic cells or may be grown by any other means available to thoseof skill in the art. Chimeric plants comprising transgenic cells arealso provided.

The expression of the polypeptide and the polynucleotides encoding thepolypeptides in the transgenic plant is altered relative to the level ofexpression of the native polypeptides in a control soybean plant. Inparticular the expression of the polypeptides in the root of the plantis increased. The transgenic plant has increased resistance to nematodesas compared to the control plant. The transgenic plant may be generatedfrom a transgenic cell or callus using methods available to thoseskilled in the art.

The Examples provided below are meant to be illustrative and not tolimit the scope of the invention or the claims. All references andappendices cited herein are hereby incorporated by reference in theirentireties.

EXAMPLES

To identify the gene conferring resistance to SCN in the PI88788 soybeanwithin the locus identified by Kim et al., 2010 (within the chromosomalinterval defined by the termini BARCSOYSSR_(—)18_(—)0090 andBARCSOYSSR_(—)18_(—)0094), a candidate gene testing approach was used.This approach is described in Melito et al. (BMC Plant Biology 2010,10:104), which is incorporated herein by reference in its entirety.Briefly, this candidate gene approach was completed with various genesat the Rhg1 locus defined above using a resistant soybean varietyFayette, which carries the PI88788-derived rhg1-b allele of the Rhg1locus, to make transgenic soybean roots that early gene-silencingconstructs and then testing these transgenic roots for loss of SCNresistance. The silencing strategy used is depicted in FIG. 2. Theartificial microRNA used in the Melito et al. reference was replacedwith artificial microRNA sequences directed against various candidate orputative genes within the Rhg1 locus. The expression of the artificialmicroRNAs was driven by the soybean Ubi3 promoter. The construct alsocontained a GFP reporter such that transformed roots could readily beidentified by GFP expression. Transgenic soybean roots expressingartificial micro-RNA (amiRNA) or hairpin (RNAi) constructs were producedusing Agrobacterium rhizogenes. Roots expressing GFP were selected forfurther analysis. Transgenic roots were inoculated with SCN to test fordecreased or increased resistance to SCN caused by candidate genesilencing conditioned by artificial microRNA expression.

Soybean resistance to SCN was measured two weeks after root inoculationby determining the proportion of the total nematode population that hadadvanced past the J2 stage in each root (FIG. 3A), relative to knownresistant and susceptible controls. Silencing any of three closelylinked genes, namely Glyma18g2580, Glyma18g2590, and Glyma18g2610, atthe rhg1-b locus of the SCN-resistant soybean variety Fayettesignificantly reduced SCN resistance (FIG. 3B). Depletion of resistancewas dependent on target transcript reduction (FIG. 4). Silencing ofother genes in and around the locus did not impact SCN resistance (e.g.,FIG. 3B, genes Glyma18g02570 and 2620).

The predicted Glyma18g02610 protein product contains a Wound-inducedprotein domain (Pfam domain PF07107; M Punta, et al., (2012) The Pfamprotein families database. Nucleic Acids Research Database Issue40:D290-D301 and Logemann et al., (1988) Differential expression ofgenes in potato tubers after wounding. Proc Natl Acad Sci USA 85:1136-1140) and a homologous (55% identical) protein in ice plant(Mesembryanthemum crystallinum) was previously shown to be responsive toboth biotic and abiotic stimuli (Yen et al., Environmental anddevelopmental regulation of the wound-induced cell wall protein WI12 inthe halophyte ice plant. Plant Physiol. 127:517-528). The annotatedprotein product of Glyma18g02610 does not have other widely knownprotein domains or inferred biochemical functions that, at the presenttime, are obvious to those with normal skill in the art. However, theabove results indicate that Glyma18g02610 is necessary for fullRhg1-mediated SCN resistance.

A Genomic Duplication of Four Genes at Rhg1 in Glycine max is Present inthe Tested SCN-Resistant Lines

Concurrent study of the physical structure of the rhg1-b locus revealedan unusual genomic configuration. A 31.2 kb genome segment, encoding theabove three genes that contribute to SCN resistance, is present inmultiple copies in SCN resistant lines (FIGS. 5, 6). The DNA sequence offosmid clone inserts carrying genomic DNA from the rhg1-b geneticinterval identified a unique DNA junction, not present in the publishedWilliams 82 soybean genome, in which a 3′ fragment of Glyma18g02570 isimmediately adjacent to the intergenic sequence downstream of(centromeric to) Glyma18g02610 (FIG. 5A). The genomic repeat containsfull copies of Glyma18g02580, -2590, -2600 and -2610 as well as thefinal two exons of Glyma18g02570. Whole-genome shotgun sequencing of aline containing rhg1-b revealed ten-fold greater depth of coverage ofthis interval relative to surrounding or homologous regions (FIG. 5B),suggesting the presence of multiple repeats.

Sequencing and PCR amplification confirmed the presence of theGlyma18g02610-2570 junction in DNA from multiple SCN-resistant soybeanaccessions, including accessions that carry the commercially importantPI 88788, Peking and PI 437654 haplotypes of the Rhg1 locus (FIG. 5C andFIG. 7). The junction was not detected in four tested SCN-susceptiblevarieties including Williams 82 (FIG. 7B). This constitutes a directtest for economically desirable alleles of the Rhg1 locus. The sharedidentity of the junction sites from disparate sources of SCN resistancesuggests a shared origin of the initial resistance-conferring event atRhg1.

Fiber-FISH (fluorescence in situ hybridization) was utilized to directlydetermine the number of copies and arrangement of the 31 kb repeatsegment in different haplotypes of the Rhg1 locus. The hybridizationpattern and DNA fiber length estimates generated using these probes(FIG. 6 and Table 1) are consistent with the presence of a single copyof the repeat in Williams 82, as in the reference soybean genome. InFayette, fiber-FISH revealed ten copies per DNA fiber of the predicted31 kb repeat segment, in the same configuration throughout the multiplenuclei sampled, in a pattern indicating ten direct repeats abutting in ahead-to-tail arrangement (FIG. 6 and Table 1). No additional copies(e.g., at other loci) were evident. In samples from soybean line Peking,three copies per DNA fiber were present in apparent direct repeatorientation (FIG. 6). Although fiber-FISH cannot resolve small sequencedifferences, the single size of all junction-amplification PCR productsand the consistency of all junction sequences assembled from fosmid orgenomic DNA sequencing (FIG. 7) further suggest the presence of adjacentdirect repeat copies.

To discover additional copy number and DNA polymorphisms we analyzedwhole genome re-sequencing (WGS) data for 41 soybean lines, the “NAMparents” from a current nested association mapping study, using sequencedata provided by Dr. Perry Cregan. The data set consists of whole genomeshotgun sequencing reads produced using Illumina Hiseq equipment andprotocols, with average depth of sequencing coverage ranging from 5 to60 fold. Reads were mapped to the Williams 82 reference genome(Phytozome, assembly 189) and analyzed for read depth (RD), INDELs andSNPs relative to the SCN susceptible line Williams 82. When thisIllumina read depth was used to estimate copy number at the Rhg1 locus,using methods analogous to those used to generate FIG. 5B, 8 of the 41soybean lines analyzed had a normalized read depth for the approximately31 kb Rhg1 region (corresponding to the rhg1-b repeat segment describedabove) that differed by greater than 5 standard deviations of the meanfrom the read depth of the two 30 kb regions immediately flanking theregion corresponding to the rhg1-b repeat segment. See Table 2. Seven ofthose lines had an estimated copy number ranging from 9.2 to 9.9 copies.These lines have PI 88788 in their pedigree, where pedigree areavailable, and have been classified as SCN-resistant in laboratoryand/or field tests. The other genotype predicted to carry Rhg1 repeatshad an estimated copy number of 2.9. Its pedigree contains both Pekingand PI 437654. The Rhg1 loci derived from Peking and PI 437654 arewidely recognized to be much less effective at conferring SCN resistanceif they are not coupled with preferred alleles of an unlinked locus,Rhg4. All other genotypes (33) were estimated to contain one copy of theapproximately 31 kb of Rhg1 DNA described in this document. All twelveof the 33 lines that had an available, previously determined SCNresistance phenotype were listed as SCN susceptible, while informationon the SCN resistance phenotype was not readily available for the other21 lines. As a control, read depth was used to estimate copy number atthe homologous region on Chromosome 11. The estimated copy number wasapproximately 1 in all tested genotypes. In addition to those linesshown in Table 2, we recently determined that a variety known as Cloud(PI 548316), that displays intermediate levels of SCN resistance,carries seven copies of the Rhg1 locus repeat segment.

The source of the first duplication event to arise at Rhg1 is not known,but was possibly the result of nearby Ty1/copia-like retrotransposonRTvr1 or RTvr2 activity. Later copy number expansion may have occurredby rare unequal exchange events between homologous repeats duringmeiotic recombination.

Genes within the Duplicated Gene Block at rhg1-b are Expressed at HigherLevels than their Homologs from SCN-Susceptible Rhg1 Haplotypes

Gene expression analysis using quantitative PCR (qPCR) determined thatthe three genes found to impact SCN resistance exhibit significantlymore transcript abundance in roots of SCN-resistant varieties relativeto susceptible lines (FIG. 5D and FIG. 7D). In contrast, the transcriptabundance for genes immediately flanking the SCN-impacting genes did notdiffer significantly between SCN-resistant and susceptible varieties(FIG. 5; Glyma18g02600 expression in roots is at or below the limits ofdetection of qPCR, cDNA cloning and RNAseq methods; See Cook et al. 2012methods and Severin et al. RNA-Sect Atlas of Glycine max: A guide to thesoybean transcriptome. Bmc Plant Biology, 2010). Full-length transcriptswere confirmed for Glyma18g02580, -2590 and -2610, and no hybridrepeat-junction transcripts were detected for Glyma18g02570 (FIG. 7E).The above suggested that elevated expression of one or more of theSCN-impacting genes could be a primary cause of elevated SCN resistance.

Quantitative real-time PCR (qPCR) was also used to examine and comparethe mRNA transcript abundance of five genes at the Rhg1 locus innon-inoculated roots of the Mg-typing soybean lines. These lines havebeen established ad accepted by researchers as representing a useful anddiverse set of SCN resistant soybean lines (Niblack et al., 2002, J. ofNemat. 34(4): 279-288; T. L. Niblack, K. N. Lambert, G. L. Tylka, 2002,Annu. Rev, Phytopathol. 44:283-303). Transcript abundance for three ofthe genes, Glyma18g02580, Glyma18g02590, and Glyma18g02610 are allexpressed more highly in each of the 7 tested SCN differentials relativeto the SCN susceptible line Williams 82 as shown in FIG. 8. Another geneat the locus, Glyma18g02600, was also more highly expressed in the SCNresistant lines, but the data for Glyma18g02600 may be less accurate andthe absolute measured transcript level of Glyma18g02600 was near thelimit of detection (consistent with published RNA-seq data from soybeanroots). As a control, a neighboring, but not duplicated gene,Glyma18g02570 shows similar expression pattern for all tested genotypes.In a separate experiment, two additional genes, (Glyma18g02620 andGlyma18g02630, flanking the repeat to the centromeric side, also showsimilar transcript abundance across SCN resistant and susceptible tines.

Four of the SCN resistant genotypes (Peking, PI 90763, PI 89772, and PI437654) are similar to each other in their level of mRNA abundance forthe four genes Glyma18g02580, Glyma18g02590, Glyma18g02610, andGlyma18g02600. In these genotypes, transcript abundance is 1.5 to 5 foldhigher for the four repeated genes relative to Williams 82. Separately,the SCN-resistant genotypes Cloud, PI 88788, and 209332 are similar toeach other in their levels of elevated mRNA abundance for Rhg1 genescompared to Williams 82 and the previous four genotypes. Transcriptabundance ranged from 4 to 20 fold higher for the repeated genes in theCloud, PI 88788, and PI 209332 genotypes. These data show that theincreased DNA copy number encoding these four genes increases thetranscript abundance. There is also a strong grouping for DNA copynumber and transcript abundance, making at least two classes withgenotypes Peking, 90763, 89772, and 437654 together, and genotypesCloud, PI 88788, and 209332 together. We note the correlation of theseRhg1 genotype (copy number) andGlyma18g02580/Glyma18g02590/Glyma18g02610 expression level groupingswith the SCN resistance phenotype groupings reported by Colgrove et al.2008 (Colgrove, A. L., and T. L. Niblack. 2008. Correlation of femaleindices from virulence assays on inbred lines and field populations ofHeterodera glycines. Journal of Nematology 40:39-45). WhileGlyma18g02600 is more highly expressed in SCN resistant lines, there isnot a clear relationship between copy number and transcript abundance asfound for the other three genes in the repeat. This suggests thatincreased DNA content does increase transcript abundance, but not in adosage dependent fashion for Glyma18g02600.

Rhg1 DNA Methylation State is Cultivar-Dependent for Genes within theDuplicated Gene Block.

To address the mechanism leading to the observed higher gene expressionin SCN-resistant cultivars, we assessed DNA methylation of the Rhg1locus using methylation sensitive restriction enzyme digestion and PCR.McrBC is an endonuclease that specifically cleaves DNA containing5-methylcytosine (5-mC) while leaving un-methylated DNA intact. DNAincubated with McrBC and then subjected to PCR fails to produce aproduct if the product spans methylated cytosines. We identifiedsignificant and reproducible differences between SCN-resistant andSCN-susceptible cultivars when soybean genomic DNA was tested formethylation at the Rhg1 locus. For example, three different primer pairsfor the Glyma18g02610 promoter or coding regions either failed toamplify a product, or the product was greatly reduced, betweenMcrBC-digested and undigested genomic DNA from resistant cultivars (FIG.9). The same primer pairs used for PCR with DNA collected fromsusceptible cultivars produced similar products whether the genomic DNAtemplate had been digested or not, indicating little or no DNAmethylation.

Interestingly, a consistent correlation between hypermethylated DNA andelevated gene expression was discovered in the SCN-resistant cultivarstested. The promoter region for Glyma18g02580, Glyma18g02590 andGlyma18g02610 were all methylated and showed higher transcription. Aneighboring gene, Glyma18g02620, did not display polymorphic methylationor altered gene expression between resistant and susceptible cultivars(FIG. 9).

Further Characterization of 2580, 2590 and 2610 Genes

RACE PCR for Glyma18g02590 and Glyma18g02610 from Fayette (notinoculated with SCN) revealed that the transcripts derived from theSCN-resistant PI88788 haplotype have identical start and stop sites tothe annotated transcripts associated with the Williams 82(SCN-susceptible) genome sequence that is available at Phytozome(http://www.phytozome.net/cgi-bin/gbrowse/soybean/). As an initial testfor readily detectable protein degradation or post-translationalmodification differences between SCN-resistant as opposed toSCN-susceptible soybean lines (not inoculated with SCN), proteinimmunodetection experiments by western blot using 1.5 kb of Fayettenative promoter driving Glyma18g02590-HA (2590_(Fayette)::2590-HA) orusing 3.2 kb of Fayette native promoter driving Glyma18g02610-HA2610_(Fayette)::2610-HA) revealed a detectable protein product thatmigrated at approximately the predicted size for the respectiveproteins, and did not reveal protein size differences in Williams 82 asopposed to Fayette (FIG. 10).

To explore the possibility that impacts of Glyma18g02580, Glyma18g02590and Glyma18g02610 on SCN development in soybean correlate withSCN-inducible gene expression, we analyzed gene expression in excisedroot tissue from heavily SCN-infested root segments of resistant andsusceptible lines. Modest increases in the expression of Glyma18g02580and Glyma18g02610 were observed in SCN-resistant Fayette, above the highlevels of expression already present in non-inoculated Fayette comparedto SCN-susceptible Williams 82 (FIG. 11).

To further explore the possibility that impacts of Glyma18g02580,Glyma18g02590 and Glyma18g02610 on SCN development in soybean rootscorrelate with SCN-inducible expression, promoter-GUS fusion constructs(Prom₂₅₈₀::GUS, Prom₂₅₉₀::GUS, Prom₂₆₁₀::GUS) were made and expressed intransgenic roots. Transgenic roots were stained for GUS activity 5 daysafter inoculation with SCN (FIG. 12). Prom₂₅₈₀::GUS roots had a moderatelevel of background staining and appeared to have brighter staining ofswollen plant vascular tissue at the head of infecting J2 SCN (apparentdeveloping syncytia). Prom₂₅₉₀::GUS roots showed very low levels ofbackground staining and consistently greater GUS staining at apparentsyncitia, along with strong staining of the tips of emerging lateralroots. Prom₂₆₁₀::GUS roots had very high levels of background stainingbut also appeared to be more highly stained at areas likely to formsyncytia. The levels of elevated GUS staining in apparent syncitia weresimilar to those we observed in positive control promoter-GUSexperiments using the previously characterized syncitium-induciblepromoter for Glyma14g06080. As noted above, the predicted Glyma18g02610encoded protein contains a Wound-Induced protein domain (Pfam07107) anda homologous (55% identical) protein in ice plant (Mesembryanthemumcrystallinum) was shown to be responsive to both biotic and abioticstimuli. Non-transgenic Fayette soybean plants exposed to MeJA hadsignificantly elevated Glyma18g02610 transcript abundance compared toneighboring genes, further documenting stress-associated expression ofthis gene (FIG. 11).

Amino acid polymorphism or overexpression of any one of the threeidentified rhg1-b genes did not account for SCN resistance on its own.From all available rhg1-b sequence reads (across multiple repeatcopies), no predicted amino acid polymorphisms relative to Williams 82were identified for Glyma18g02580, Glyma18g02600 or Glyma18g02610. Somecopies of Glyma18g02590 from rhg1-b resemble the Williams 82 sequence,while others contain a set of polymorphisms, notably at the predictedC-terminal six amino acids of the predicted α-SNAP protein (Table 3,confirmed by cDNA sequencing).

The whole-genome sequencing (WGS) data were also analyzed for DNApolymorphisms such as insertions or deletions (INDELs) and singlenucleotide polymorphisms (SNPs). In the seven genotypes with anestimated copy number ranging from 9 to 10, a number of SNPs wereidentified relative to the reference Williams 82 sequence. One genecontained in the repeated rhg1-b segment of DNA, Glyma18g02590, containsDNA polymorphisms relative to Williams 82 as defined above. There is asingle SNP, C to A (Williams 82 to PI 88788 derived), at position1,643,192 that results in a Q to K amino acid substitution. There are 3SNPs present at the C-terminus, occurring at positions 1,644,965 (G toC), 1,644,968 (G to C), and 1,644,974 (C to A), and a 3 bp insertionafter base 1,644,972 (GGC) that collectively change the final 5 aminoacids of the Williams 82 protein from EEDLT to QHEAIT. The nucleotideand amino acid sequences are shown in FIG. 13. The 3 bp insertion causesan extra amino acid in the PI 88788 derived lines. All base pairpositions correspond to the Williams 82 genome version 1.1, assembly189. Numerous SNP and INDEL, polymorphisms were observed within theapproximate 31 kb Rhg1 repeat DNA region, between Williams 82 and PI88788-source Rhg1, in the nucleotide regions outside of those thatdirectly comprise the final open reading frame of Glyma18g02580,Glyma18g02590, Glyma18g2600, and Glyma18g02610. Analyses of Illuminasequencing read depth, in the seven soybean lines from the NAMsequencing project with an estimated copy number ranging from 9 to 10,indicated that there were 9 very similar copies of the PI 88788-typerepeat at rhg1-b, and one partial copy of a Williams 82-like repeat atrhg1-b.

The soybean line LD01-5907 from the soybean NAM parent sequencingproject, which carries an estimated copy number of 3, also contains DNApolymorphisms affecting the amino acid sequence for Glyma18g02590. TheDNA polymorphisms are different than those found in PI 88788 derivedlines, but occur at similar positions. There is a SNP at position1,643,225 that results in a D to E amino acid substitution. There are 2SNPs present at the C-terminus, occurring at positions 1,644,968, (G toT) and 1,644,974 (C to A) and a 3 bp insertion after base 1,644,972(GGT) that collectively change the final 5 amino acids of the Williams82 protein from EEDLT to YEVIT. The 3 hp insertion causes an extra aminoacid in the Glyma18g02590 protein product in lines with an Rhg1 locusderived from Peking or PI 437654 sources.

The DNA polymorphisms for Glyma18g02590 identified through WGS analysiswere confirmed to be expressed using 3′ RACE and (DNA sequencing. In SCNresistant genotypes Cloud, PI 88788, and PI 209332, two differentGlyma18g02590 transcripts were identified. One of the sequencescorresponded to the Williams 82 reference type sequence, and the othercorresponded to the sequence from PI 88788-derived resistant sources(from NAM parents). The proportion of PI 88788-derived versus Williams82-type cDNA sequence follows that observed for DNA sequence. That is,the cDNA of PI 88788 derived Glyma18g02590 is roughly 90% of the totaltranscripts sequenced. This is consistent with the data that thesegenotypes contain 8 or 9 copies of the 31 kb DNA segment derived from PI88788. A SNP present in the 5′ UTR of Glyma18g02610 was also analyzed inPI 88788. The proportion of the sequence types fits the otherObservations.

In SCN-resistant genotypes Peking, PI 90763, PI 89772, and PI 437654 twodifferent transcripts were identified for Glyma18g02590. One of thesequences corresponds to the sequence from the Peking/PI 437654-derivedresistant source LD01-5907 from the NAM sequencing project. See FIG. 13.An alternative form of cDNA was also detected from each of the fourSCN-resistant genotypes Peking, PI 90763, PI 89772, and PI 437654, withthe same type of polymorphism across all four sources. This apparentmRNA splicing isoform had 36 nucleotides deleted resulting in aGlyma18g02590 isoform with 12 fewer amino acids as shown in FIG. 13. Thedeletion occurs at the end of exon 6 and splices back into frame in exon7. None of the sequenced products from Peking, PI 90763, PI 89772, andPI 437654 contained the Williams 82-type Glyma18g02590 sequence,consistent with the WGS analysis. Based on the proportion of cDNAssequenced, very approximately 70% to 90% of the Glyma18g02590 transcriptis the full-length version in these lines.

The various polymorphisms may result in functional differences in theGlyma18g2590 polypeptide and are modeled three-dimensionally in FIG. 14which relies on the solved crystal structure of the yeast Sec17 protein.The deleted alpha-helix is shown in tight gray. It is noted that thesepolymorphisms are clustered in one general area near the C-terminus ofthe predicted folded Glyma18g02590, which is an alpha-SNAP proteinhomolog, and that substantial functional data are available foreukaryote alpha-SNAP proteins that suggest particular functions for thisregion of the protein (e.g., Barnard R J, Morgan A, & Burgoyne R D(1996) Domains of alpha-SNAP required for the stimulation of exocytosisand for N-ethylmalemide-sensitive fusion protein (NSF) binding andactivation. Molecular biology of the cell 7(5):693-701; Barnard R J,Morgan A, & Burgoyne R D (1997) Stimulation of NSF ATPase activity byalpha-SNAP is required for SNARE complex disassembly and exocytosis. JCell Biol 39(4):875-883; Jahn R & Scheller R H (2006) SNAREs—engines formembrane fusion. Nat. Rev. Mol. Cell Biol. 7(9):631-643.)

However, expressing only the Fayette polymorphic rhg1-b-typeGlyma18g02590 downstream of a strong constitutive promoter or nativepromoter sequence did not increase the SCN resistance reaction ofWilliams 82 transgenic roots (FIG. 15 and FIG. 16), suggesting thatrhg1-b SCN resistance requires more than this 2590 amino acidpolymorphism. But such polymorphisms may play a contributing rote SCNresistance that was not detected in these experiments. Overexpression ofGlyma18g02580 or Glyma18g02610 also failed to increase SCN resistance(FIG. 13) when expressed alone using a strong constitutive promoter.These data are preliminary and may indicate that the resistancephenotype requires more than a single gene or that some other factor isnecessary to mediate resistance.

Given the above, simultaneous overexpression of the set of genes withinthe 31 kb repeat segment was tested as a possible source of SCNresistance. A single recombinant DNA construct was made in which each ofthe genes Glyma18g02580, Glyma18g2590, Glyma18g2600 and Glyma18g2610 wasfused to a strong promoter. In two separate experiments that togethertested >25 independent transgenic events for each DNA construct,resistance to SCN was significantly increased in SCN-susceptibleWilliams 82 by simultaneous overexpression of this set of genes (FIG.15). Increased SCN resistance was conferred despite the fact that threeof the genes being overexpressed encode predicted amino acid productsidentical to those of SCN-susceptible Williams 82, and the polymorphicFayette rhg1-b Glyma18g02590 gene that was used was not sufficient tocause a detectable change in SCN resistance when overexpressed on itsown (FIG. 15). Of note, there was no significant elevation of PR-1 inthese transgenic roots, which could have indicated non-specificelevation of defenses (FIG. 16). We also tested the impact ofsimultaneously over-expressing Glyma18g02580, Glyma18g02590_(Fayette),and Glyma18g02610 on SCN resistance in Williams 82. We observedincreased resistance to SCN transgenic roots of Williams 82over-expressing Glyma18g02580, Glyma18g02590_(Fayette), andGlyma18g02610 relative to Williams 82 empty vector roots as shown inFIG. 17. These data indicate that over-expressing this combination ofgenes results in enhanced SCN resistance.

These results reveal a novel mechanism for disease resistance: anexpression polymorphism for multiple disparate but tightly linked genes,derived through copy number variation at the Rhg1 locus. This knowledgesuggests future approaches to enhance the efficacy of Rhg1-mediatedquantitative resistance to the highly important SCN disease of soybean,for example through isolation of soybean lines that carry more copies ofthe 31 kb Rhg1 repeat, or through transgenic overexpression of therelevant genes. These approaches may be applicable in other species aswell, for resistance to other endoparasitic nematodes.

The biochemical mechanisms of Rhg1-mediated resistance remain unknown.Other sequenced plant genomes do not carry close homologs of thepredicted Glyma18g02610 protein, although a wound-inducible protein inice plant with 55% identity has been studied. Modeling of theGlyma18g02610 predicted tertiary structure using Phyre2 indicated, with98% confidence, similarity of 48% of Glyma18g02610 to the PhzA/Bsubfamily of Delta(5)-3-ketosteroid isomerase/nuclear transport factor 2family proteins. Hence Glyma18g02610 may participate in the productionof phenazine-like compounds that are toxic to nematodes. Thusapplication of Glyma18g2610 to plants, soil or seeds may inhibitnematodes in susceptible plants. Secretion of the Glyma18g02610 proteinor other plant products that contribute to disease resistance may beimpacted by the Glyma18g02590 α-SNAP protein. Because it is one of atleast five α-SNAP homologs encoded in the soybean reference genome,Glyma18g02590 may have undergone subfunctionalization orneofunctionalization. Fully sequenced plant genomes carry from two dozento over five dozen annotated amino acid transporters of many subtypes(www.phyotzome.net), which can be involved in amino acid import and/orexport between cells or between subcellular organelles. TheGlyma18g02580 protein and its most closely related transporters ofsoybean and other species are not functionally well-characterized, sothe concept that Glyma18g02580 alters nematode success by altering thelevels of specific amino acids or amino acid derivatives at the feedingsite is only one of many viable hypotheses for future study regardingthe SCN-deterring function of Glyma18g02580.

Copy number variation (CNV) of a block of dissimilar genes, rather thanCNV for a single gene family, confers Rhg1-mediated SCN resistance.Recent analyses of genome-architecture in sorghum, rice, and soybeanhave reported high levels of CNV, and a tendency for overlap of regionsof CNV with postulated biotic and abiotic stress-related genes. Thepresent work provides a concrete example of CNV conferring a valuabledisease resistance trait. In humans and insects, adaptive traits havebeen associated with CNV for specific single genes. Single-copy clustersof functionally related but non-homologous genes are highly unusual inmulticellular eukaryotes, but these have been reported in associationwith plant secondary metabolism. We provide a unique example of CNVinvolving more than two repeats, with the repeat encoding multiple geneproducts that are necessary for adaptation to the same importantenvironmental constraint. Given the highly repetitive nature andplasticity of plant genomes and the relatively underexplored associationbetween CNV and phenotypes, it seems likely that a number of othercomplex traits are controlled by the general type of CNV we report forsoybean Rhg1.

Materials and Methods:

Agrobacterium rhizogenes Soybean Root Transformation

A. rhizogenes strain Arqua1 was transformed by freeze-thaw as previouslyreported by Wise, A. A., Z. Liu, and A. N. Binns, Three methods for theintroduction of foreign DNA into Agrobacterium. Methods Mol Biol, 2006,343: p, 43-53 and Hofgen, R. and L. Willmitzer, Storage of competentcells for Agrobacterium transformation. Nucleic Acids Research, 1988.16(20): p. 9877-9877. The cells were plated on selective media with theappropriate antibiotic and incubated at 28° C. for two days. A.rhizogenes strain Arqua1 was received from Dr. Jean-Michel Ane,University of Wisconsin Madison. Soybean seeds lacking macroscopic signsof fungal or viral contamination were surface-sterilized for 16-20 h ina desiccator jar with chlorine gas generated by adding 3.5 ml 12N HClinto 100 ml household bleach (6% sodium hypochlorite). At least 20 seedsper experiment were plated onto germination media (Gamborg's B5 salts(3.1g/L), 2% sucrose, 1× Gamborg's B5 vitamins, 7% Noble agar, pH 5.8)in 100×25 mm Petri plates. Plates were wrapped with Micropore tape (3M,St. Paul, Minn.) and incubated at 26° C. in a growth chamber (18/6light/dark hours) for approximately one week. Soybean cotyledons wereharvested 5-7 days after germination by gently removing them from thehypocotyls with sterile forceps. With a sterile forceps and Falcon #15scalpel, several shallow slices were made across the abaxial surface ofthe cotyledons after dipping the scalpel in A. rhizogenes suspension(OD₆₀₀ 0.6-0.7 in sterile ddH₂0). The cotyledons were then placedabaxial-side down on a co-culture medium (CCM) (0.31 g/L Gamborg's B5salts, 3% sucrose, 1× Gamborg's B5 vitamins (BioWorld, Dublin Ohio), 0.4g/L L-cysteine, 0.154 g/L dithiothreitol, 0.245 g/L sodium thiosulfate,40 mg/L acetosyringone, 5% Noble agar, pH 5.4) in 100×15 mm Petri plateswith a piece of 70 min filter paper (Whatman, Piscataway, N.J.) on thesurface of the agar to prevent A. rhizogenes from overgrowing. Plateswere wrapped with parafilm and incubated in the dark at room temperaturefor three days. The explants were then transferred to a hairy rootmedium (HRM) of 4.3 g/L MS salts (Sigma Co., St. Louis, Mo.), 2%sucrose, 1× Gamborg's 85 vitamins (BioWorld, Dublin, Ohio), 7% Nobleagar, 0.15 g/L cefotaxime, 0.15 g/L carbenicillin, pH 5.6 in 100×15 minPetri plates, wounded side up. Plates were wrapped with Micropore tapeand incubated in the dark at room temperature until roots emerged,usually in around 2 weeks. Transgenic soybean roots were detected basedon plasmid vector-encoded GFP expression, using a fluorescencestereomicroscope (LEICA MZ FL III with GFP2 filter). Transgenic soybeanroot tip segments (2-3 cm were transferred to HRM. Roots that wereexpressing incomplete strips of fluorescence (chimeras) or exhibitingoverall low levels of GFP fluorescence were avoided. Independenttransgenic events, generated from different inoculation sites ordifferent cotyledons, were maintained separately for RNA extraction andnematode demographic assays.

Nematode Maintenance

An SCN population from Racine, Wis. (Hg type 7), collected by AnnMacGuidwin (University of Wisconsin-Madison), was maintained on thesusceptible soybean cultivar Williams 82. Seeds were germinated betweentwo damp pieces of paper towel that were rolled-up and placed verticallyin a glass beaker with a small amount of water at the bottom for 2-4days. Germinated seeds were then planted in autoclaved 4:1 sand:soilmixture and inoculated with 2000 eggs of a H. glycines per plant, andgrown in a 28° C. growth chamber. Cysts were collected ˜50 days afterinfection when soybeans were at R2 (full (lowering) and extracted fromsoil and roots using sieves and centrifugation. Briefly, soil and rootsfrom infected pots was placed in a pitcher of water and agitated. Thesoil-cyst-water slurry was passed over a 710 μm-250 μm sieve tower, andthe mixture from the 250 μm sieve was backwashed into a 50 mL plasticconical tube. The tubes were centrifuged at 2000 rpm for 4 minutes thenthe supernatant was poured off. A 60% sucrose solution was added to thetubes, stirred, and centrifuged at 2000 rpm for 2 mM. Cysts in thesupernatant were then collected over a 250 μm sieve. Collected cystswere stored at 4° C. sealable plastic bags containing twice-sterilizedflint sand.

Nematode Demographics Assay

Nematode demographics assays were performed as in Melito et al, infra.Vigorous new root segments (2-3 cm including root tip) were utilized.All roots (all genotypes within an experiment) were coded with a randomnumber prior to inoculation, to mask root genotype information from theinvestigators who stained roots two weeks later and determined thenumber of nematodes in each nematode development category. For inoculum,H. glycines eggs were collected by breaking open cysts with a largerubber stopper and collecting the eggs on a sieve stack consisting of250 μm-75 μm-25 μm sieves (USA Standard Testing Sieve). Eggs werecollected from the 25 μm sieve and rinsed. Eggs were placed in a hatchchamber with 3 mM ZnCl₂ for hatching at room temperature in the dark for5-6 days. See Wong, A. T. S., G. L. Tylka, and R. G. Hartzler, Effectsof 8 herbicides on in-vitro hatching of Heterodera-glycines. Journal ofNematology, 1993. 25(4): p. 578-584. Hatched 12 nematodes weresurface-sterilized for 3 min in 0.001% mercuric chloride and washedthree times with sterile distilled water, then suspended in roomtemperature 0.05% low-melting point agarose to facilitate evendistribution. Baum, T. J., M. J. E. Wubben, K. A. Hardy, H. Su, and S.R. Rodermel, A screen for Arabidopsis thaliana mutants with alteredsusceptibility to Heterodera schachtii. Journal of Nematology, 2000.32(2): p. 166-173. The number of active nematodes was determined byviewing an aliquot under a stereomicroscope at least one-half hour aftersurface-sterilization and washing, and 200-250 active J2s wereinoculated onto each fresh root segment. Inoculated roots with nematodeswere maintained on HRM media, at 28° C.; substantial root growthtypically occurred during the subsequent two weeks. Nematode infectionand development within these root systems was monitored by clearing andstaining with acid fuchsin, typically 15 days post inoculation (dpi),Bybd, D. W., T. Kirkpatrick, and K. R. Barker, An improved technique forclearing and staining plant-tissue for detection of nematodes. Journalof Nematology, 1983. 15(1): p. 142-143. The nematode demographic assaywas then completed by recording the number of nematodes in each rootsystem that exhibited a morphology resembling either J2 (thin), J3(sausage-shaped), elongated male, or J4/adult female nematodes, as notedin text and figures. Typically, 20-80 nematodes were present in eachroot; roots containing fewer than ten nematodes were excluded fromfurther analysis. Results were expressed as % of nematodes that haddeveloped beyond J2 stage ([J3+adult males+adult females]/[J2+J3+adultmales+adult females]). Each data point was normalized to the mean forWilliams 82 roots transformed with empty vector, from the sameexperiment. All reported data are based on at least two independentbiological replicate experiments 12 independently transformed roots foreach bar on a bar graph).

Primer Table

Primer sequences used to perform this research are listed in Table 4 andreferred to by number in this document.

Vector Construction for Soybean Transformation

Binary vectors pSM101 and pSM103 for soybean transformation wereconstructed as previously described in Melito et al. To generate andclone soybean amiRNAs, the Web microRNA Designer(http://wmd3.weigelworld.org) and protocols were used. The concept ismore thoroughly documented in other references. See Schwab, R., S.Ossowski, M. Riester, N. Warthmann, and D. Weigel, Highly specific genesilencing by artificial microRNAs in Arabidopsis, Plant Cell, 2006.18(5): p. 1121-1133. Soybean DNA was extracted from either expandingsoybean trifoliates or soybean roots using a previously reported CTABmethod. Doyle, J. J. and E. E. Dickson, Preservation of plant-samplesfor DNA restriction endonuclease analysis. Taxon, 1987. 36(4): p.715-722. PCR fragments for amiRNA construction were TA cloned usingpCR8/GW/TOPO TA cloning kit (Life Technologies Corp., Carlsbad Calif.)(Table 4 13-24). Binary vectors pGRNAi1 and pGRNAi2 for soybeantransformation were a gift from Wayne Parrot, University of Georgia(unpublished). For each hairpin, a 300-600 bp DNA fragment was PCRamplified (Table 4 1-12) using Phusion HF polymerase (New EnglandBiolabs, Ipswich, Mass.) and iScript cDNA synthesis kit (Biorad,Hercules, Calif.) as a template, as per manufacturer's instructions. PCRproducts were TA cloned as previously described. Primers used togenerate the DNA fragments were designed to contain restriction sitesAvrII/AscI (forward primer) and BandHI/SwaI (reverse primer) to allowcloning into pGRNAi1 and pGRNAi2. To generate the first arm of thehairpin, the insert and vector were sequentially digested withrestriction endonucleases SwaI and AscI using manufacturer's recommendedprotocol (New England Biolabs, Ipswich, Mass.). DNA was separated on a1.0% agarose gel stained with ethidium bromide, and respective DNAfragments were gel purified using Qiaquick gel extraction kit (Qiagen,Valencia, Calif.) and ligated together overnight at 4° C. using T4 DNAligase (Promega, Madison, Wis.). The same procedure was used to insertthe second arm of the hairpin construct using the restrictionendonucleases BandHI and AvrII. To construct single gene overexpressionvectors for Glyma18g02580 (Table 4 70, 71), Glyma18g02590 (Table 4 57,58) and Glyma18g02610 (Table 4 55, 56), full-length ORFs were PCRamplified from cDNA Fayette using Phusion HF polymerase and TA cloned inpCR8/GW/TOPO as previously described. Glyma18g02600 (Table 4 67, 68) wascloned from genomic DNA by similar methods, as no Glyma18g02600 cDNAcould be detected in root cDNA libraries. The Glyma18g02610 andGlyma18g02590 ORFs were recombined with pGWB14 (CaMV 35S promoter, 6XHA-NOS terminator) using LR clonase reaction (Life Technologies Corp.,Carlsbad, Calif.) per manufactures instructions. See Nakagawa, T., T.Kurose, T. Hino, K Tanaka, M. Kawamukai, Y. Niwa, K. Toyooka, K.Matsuoka, T. Jinbo, and T. Kimura, Development of series of gatewaybinary vectors, pGWBs, for realizing efficient construction of fusiongenes for plant transformation, J Biosci Bioeng, 2007. 104(1( ) p.34-41. Glyma18g02610 (Table 4 55, 59) was PCR amplified from pGWB14 andTA cloned into pCR8. This vector and pSM103 were digested with XbaI/KpnIand ligated to yield GmUbi_(prom):2610-HA:NOS_(term) (OE:2610-HA). Thesame procedure was used for Glyma18g02590 (Table 4 57, 59), except theamplicon contained XbaI/SalI sites and was TA cloned into pCR8.25904-HA:NOS_(term) and pSM103 were digested with XbaI/SalI and ligatedto yield GmUbi_(prom):2590-HA:NOS_(term) (OE:2590-HA). The fullOE:2590-HA was also digested (XbaI/SalI) and ligated into pSM103containing OE:2610-HA to yield OE:2610-OE:2590. To generate the fourgene overexpression construct, the restriction sites Pad, PspOMI, andAscI were added to pSM101 between sites PstI/HindIII by annealing oligos(Table 4 62, 63) to generate pSM101+. The two gene overexpressioncassette (OE:2610-OE:2590) was moved to the new pSM101+ using therestriction enzymes PstI/KpnI and ligation. A Nos promoter was added toGlyma18g02600 in pCR8 using overlap PCR (Table 4 65-68) and TA clonedinto pCR8. This vector was recombined with pGWB16 (no promoter,4xMyc-NOS terminator) in an LR clonase reaction to yieldNos_(prom):2600-myc:Nos_(term)(OE:2600-myc). OE:2600-myc was PCRamplified (Table 4 66, 72) and TA cloned into pCR8, and subcloned intopSM101+ (OE:2610-OE:2590) using restriction enzymes HinIII/AscI to yieldthe three gene overexpression vector (OE:2610-OE:2590-OE:2600). A Nospromoter was added to Glyma18g02580 pCR8 using overlap PCR with primers71-74 and TA cloned into pCR8. This vector was used with pGWB16 in an LRclonase reaction to yield Nos_(prom):2580-myc:Nos_(term)(OE:2580-myc).OE:2580-myc was amplified. (Table 4 72, 75) and TA cloned, thensubcloned into the three gene overexpression vector resulting in thefour gene overexpression vector pSM101+OE:2610-OE:2590-OE:2600-OE:2580.The native Fayette Glyma18g02590 (2590_(FayP):2590_(Fay)) construct forWilliams 82 complementation was subcloned from a fosmid containing thedesired allele. A 6.5 kb DNA fragment containing the PI 88788Glyma18g02590 was isolated from a fosmid following SalI digestion andcloned into pSM101 using the SalI restriction site. This sequencecontained approximately 1 kb of 5′ regulatory DNA sequence. Anadditional 600 bp of 5′ regulatory sequence directly upstream of thesubcloned region was added to the construct by amplifying a PCR product(Table 4 79, 80) from the fosmid and inserted using the restrictionenzymes HindIII/SalI. The resulting construct contained approximately1.6 kb of naturally occurring 5′ regulatory sequence of the FayetteGlyma18g02590 allele. Vector sequences were confirmed at various stepsusing Sanger sequencing with ABI Big Dye cycle sequencing kit (dideoxychain-termination) and ABI 3730xl DNA Analyzers (Life TechnologiesCorp., Carlsbad, Calif.), using the DNA sequencing service at theUniversity of Wisconsin-Madison Biotechnology Center.

Quantitative Real Time PCR

Quantitative PCR (qPCR) was performed using either the MyIQ or CFX96real-time PCR detection system (BioRad, Hercules, Calif.). cDNA wassynthesized from RNA using iScript cDNA synthesis kit (Biorad, Hercules,Calif.) per manufactures protocol by adding 0.825 ug to 1.0 ug of RNAdepending on the experiment. Total RNA was extracted from root tissue ofconventional and transgenic soybeans. RNA was extracted fromconventional soybean plants grown in Metro mix for two weeks at 26° C.and 16 hours light prior to tissue collection. Roughly 200 mg of tissuewas collected from each plant, immediately flash-frozen in liquidnitrogen and stored at −80 C. Transgenic root material was collectedfrom roots actively growing on HRM as previously described. Roughly50-100 mg of tissue was collected from each root, flash frozen in liquidnitrogen and stored at −80 C. RNA was extracted using either the RNeasyMini Kit (Qiagen, Valencia, Calif.) or TRIzol reagent (Life TechnologiesCorp., Carlsbad, Calif.) following manufactures protocols. RNAconcentrations were determined using the NanoDrop-1000 spectophotomoter(Thermo Scientific, Waltham, Mass.). DNA was removed from RNA samplesusing either RNase-free DNase I (Qiagen, Valencia, Calif.) or DNA-free(Life Technologies Corp., Carlsbad, Calif.) following manufactureprotocols. RNA integrity was determined using the 2100 BioAnlyzer(Agilent Technologies, Santa Clara, Calif.) or 500 ng of total RNA wasrun on a 1.2% agarose gel stained with ethidium bromide and visualizedunder UV-light to ensure RNA quality following extraction. qPCRreactions were carried out using either IQ SYBR Green Supermix orSsoFast EvaGreen Supermix (Biorad, Hercules, Calif.). Primerconcentrations for all reactions were between 0.2 μM and 0.3 μM. Twotechnical replicates were run per RNA. Efficiency curves were generatedfor qPCR primer pairs using cDNA from the cultivar Fayette or Williams82 following a 3-4 step, 3-5 fold dilution. Following amplification, amelt curve program was performed. To ensure qPCR fluorescent signal wasnot the results of DNA, 100 ng of RNA extraction was added directly toIQ SYBR Green Supermix or SsoFast EvaGreen Supermix with primers. DNAcontamination was considered negligible if CT values were not detecteduntil after 32-35 cycles. A control reaction was run in parallel using aknown cDNA sample. Transcript abundance for genes at Rhg1 was measuredusing primers X-X. A total of six primer pairs were tested as referencegenes (EF1B, SKIP16, UNK2, ACT11, UNK1, TIP41) (Table 4 39-50). Hu, R.B., C. M. Fan, H. Y. Li, Q. Z. Zhang, and Y. F. Fu, Evaluation ofputative reference genes for gene expression normalization in soybean byquantitative real-time RT-PCR. Bmc Molecular Biology, 2009. 10: p. 12.Reference genes were validated using Bestkeeper analysis. Pfaffl, M. W.,A. Tichopad, C. Prgomet, and T. P. Neuvians, Determination of stablehousekeeping genes, differentially regulated target genes and sampleintegrity: BestKeeper—Excel-based tool using pair-wise correlations.Biotechnology Letters, 2004. 26(6): p. 509-515. Primer pairs SKP16 andTIP41 were selected and used in subsequent experiments. Transgenic rootsexpressing empty-vector constructs analogous to the vectors carryinggene silencing or gene expression constructs were included in theexperiments as controls and used to standardize gene expression. Resultswere considered to be at the limits of detection if CT values were >35(i.e., for Glyma18g02600 transcripts).

DNA Repeat Junction Analysis

The presence of a repeat junction was confirmed using PCR (Table 4 81,82) and soybean genomic DNA from SCN resistant cultivars Fayette,Hartwig, Newton and SCN susceptible cultivars Williams 82, Essex, Thorneand Sturdy. DNA extraction and PCR were performed as previouslydescribed. Possible impacts of retrotransposons on Rhg1 locus evolutionwere investigated by searching for sequences with similarity to knownplant retrotransposons. A 185 bp sequence with 75% identity to the 5′and 3′ tong terminal repeat (LTR) regions of Ty1/copia-likeretrotransposons RTvr1 and RTvr2 is present within 400 bp of the rhg1-bduplication junction.

Statistical Analysis

Data were analyzed by ANOVA using Minitab (v.14) with the General LinearModel and Tukey Simultaneous Test.

Fosmid Library Construction

Seed of soybean Plant Introduction (PI) 88788 was obtained from the USDAsoybean germplasm collection. Plants were grown in a growth chamber setat a photocyle of 18/6 hr (day/night), 23/20° C. (day/night), and 50%relative humidity for 1-2 weeks. Young leaf tissue was collected fromsix to 15 individuals for each line. Genomic DNA was extracted usingcetrimonium bromide (CTAB). Plant samples were ground to fine powder inliquid nitrogen, transferred to 20 ml of CTAB extraction buffer (2%CTAB, 100 mM 8 Tris pH 9.5, 1.4 M NaCl, 1% PEG 6000, 20 mM EDTA, 2%polyvinylpyrrolidone, 2.5% P-mercaptoethanol), and placed at 65° C. for1 hr. After incubation, an equal volume of Phenol:Chloroform:IsoamylAlcohol (25:24:1, pH 6.7) was added to the tube, then centrifuged at8,000 g at 10° C. for 10 min. The aqueous (top) phase was transferred toa new tube and an equal volume of chloroform:isoamyl alcohol (24:1) wasadded to the aqueous phase and centrifuged. The aqueous (top) phase wasthen transferred to a new tube and 0.7 volumes of isopropyl alcohol wasadded to the aqueous phase. After mixing well, the aqueous phase wascentrifuged and the pellet resuspended in 70% EtOH, centrifuged at 7,500g for 10 min. After centrifugation, the pellet was resuspended in 100 ulof TE (10 mM Tris pH 7.5, 1 mM EDTA). The DNA was treated with RNase Aby incubating in 20 ug/ml RNase A at 37° C. for 1 hr. The PI 88788fosmid library was constructed using the CopyControl™ Fosmid LibraryProduction Kit (Epicentre, Madison, Wis.) following the manufacturer'sprotocol. Briefly, 20 ug of the size-fractionated DNA was used forend-repair. 35-45 kb fragment pools of DNA were cloned in the pCC1FOS™Vector. Ligated DNA was packaged using the MaxPlax™ Lambda PackagingExtracts and transformed into the Phage T1-Resistant EPI 300™-T1® E.coli strain.

Fosmid Clone Sequencing and Assembly

Five candidate fosmid clones were identified by PCR-based pool screeningusing primers based on the rhg1-b interval of the Williams 82 referencesequence. Once it was confirmed that end sequences matched theanticipated region of the reference soybean genome sequence, they weresequenced using both the Roche 454/GS FLX+ system (Roche) and IlluminaMiSeq (Illumina). 1-3 ug fosmid clone DNA was used for making paired-endsequencing libraries for 454/GS FLX+. After library construction, pooledbarcoded libraries were loaded onto one lane of the sequencing flow celland sequenced. The average read length was 463 bp. The number of readsgenerated from 454/GS FLX+ is as follows: fosmid clone #1 in FIG. 2A:10,865, #2: 6,271, #3: 6,648, #4: 6,520, and #5: 9,390. The reads wereassembled using Phrap/Cross_match (www.phrap.org) and CAP3. Huang, G.Z., R. Allen, E. L. Davis, T. J. Baum, and R. S. Hussey, Engineeringbroad root-knot resistance in transgenic plants by RNAi silencing of aconserved and essential root-knot nematode parasitism gene. Proceedingsof the National Academy of Sciences of the United States of America,2006. 103(39): p. 14302-14306. For the MiSeq, 0.3-2 ug of DNA was usedfor making the sequencing library. Average DNA fragment size was 550 bp(range from 430 to 720 bp). 154 cycles from each end of the fragmentswere performed using a TruSeq SBS sequencing kit version 1 and analyzedwith Casava1.8 (pipeline 1.8). Throughout the reads, the average qualityscores for each base were over 30. The number of reads generated fromMiSeq is as follows: fosmid clone #1 in FIG. 2A: 1,067,403, #2: 814,728,#3: 1,156,784, #4: 1,091,852, and #5: 946,028. ABySS was used toassemble the reads from MiSeq. Simpson, J. T., K. Wong, S. D. Jackman,J. E. Schein, S. J. M. Jones, and I. Birol, ABySS: A parallel assemblerfor short read sequence data. Genome Research, 2009. 19(6): p.1117-1123. The result was visualized using Geneious. Homopolymericsequences and other problematic regions were manually sequenced usingSanger primer walking.

Whole-Genome Shotgun Sequencing and Read Depth in Duplicated Region

Whole-genome shotgun sequencing of a soybean breeding line LD09-15087a,a near-isogenic line (NIL) that harbors rhg1-b from PI 88788, wasconducted using Illumina technology. 1.5 ug of genomic DNA was sequencedusing the Illumina HiSeq 2000 instrument with 100 bp paired-endsequencing at the University of Illinois Biotechnology Center. The DNAfragment size for the soybean whole-genome shotgun sequencing librarywas 600 bp; the library was loaded onto one lane of a flow cell andsequenced using version 3 of sequencing kits and Casava 1.8 (pipeline1.9). 312,909,668 reads (about 28× coverage of the 1.1 gb soybeangenome) were generated with all positions having average quality scores30 or higher. To examine the depth of the coverage within the duplicatedregion, reads from the sequencing were aligned to the Glyma1 version ofthe soybean genome assembly. Novoalign (v 2.08.01)(http://www.novocraft.com) with paired end options (PE 600,120) was usedto align the reads to the reference genome. Approximately 95.1% of readswere aligned to the reference sequence. The number of reads aligned tothe target interval was counted from a BAM file using SAMtools (v0.1.18). Target interval is as follows: “Block” in FIG. 5B: a 31.2 kbregion (1,632,225-1,663,455 on chromosome 18), “Block-1”: the same sizeregion as region of interest upstream, and “Block+1”: the same sizeregion as region of interest downstream. Homeologous regions onchromosome 11 (“Block” in FIG. 5B: 37,392,345-37,434,356 bp) and 2(“Block”: 47,772,323-47,791,521 bp) were identified using BLASTN.Analogous approaches were used with the soybean NAM parent sequencedata.

Fiber-FISH

Soybean nuclei were lysed to release large chromosomal segments and, incontrast to more standard FISH methods, the chromosome segments weredecondensed to generate extended DNA fibers before fixing to microscopeslides and hybridizing to fluorescently labeled DNA probes. Young leaftissues were collected from fast growing plants of Williams 82, Peking,and Fayette. Nuclei isolation, DNA fiber preparation, and fiber-FISHwere performed following published protocols. Jackson, S. A., M. L.Wang, H. M. Goodman, and J. Jiang, Application of fiber-FISH in physicalmapping of Arabidopsis thaliana. Genome, 1998. 41(4): p. 566-72. Afosmid clone spanning an rhg1-b repeat from PI 88788 was digested usingthe exonuclease SmaI (New England Biolabs, Ipswich, Mass.). The productsof the restriction digestion were separated in a 0.7% get and isolatedusing the Qiaex II gel extraction kit (Qiagen, Valencia, Calif.), DNAprobes were labeled with either biotin-1641717P or digoxigenin-11-dUTP(Roche Diagnostics, Indianapolis, Ind.) using a standard nicktranslation reaction. The fiber-FISH images were processed with MetaImaging Series 7.5 software. The final contrast of the images wasprocessed using Adobe Photoshop CS3 software. The cytologicalmeasurements of the fiber-FISH signals were converted into kilobasesusing a 3.21 kb/μm conversion rate.

Transcript Analysis

To confirm the annotation of transcripts at Rhg1, rapid amplification ofcDNA ends (RACE) PCR was performed for Glyma18g02580 (Table 4 95),Glyma18g02590 (Table 4 87-90) and Glyma18g02610 (Table 4 91-94) usingthe SMARTer RACE cDNA kit per manufacturer protocols (ClonTech, MountainView, Calif.). Following RACE, PCR products were TA cloned intopCR8/GW/TOPO as previously mentioned. Randomly chosen colonies weresequenced (Table 4 76, 77) as described to confirm the 5′ and 3′ ends ofindividual transcripts. To detect potential transcript isoforms,northern analysis was conducted using standard methods. Probes weregenerated for Glyma18g02570 (Table 4 83, 84). Absence of truncatedGlyma18g02570 transcripts (Table 4 85, 86) derived from 31.2 kb repeatjunctions was also confirmed by PCR from cDNA, using a 2570 reverseprimer and a forward primer in the most strongly predicted exon upstreamof the repeat junction. Hebsgaard, S. M., P. G. Korning, N. Tolstrup, J.Engelbrecht, P, Rouze, and S. Brunak, Splice site prediction inArabidopsis thaliana pre-mRNA by combining local and global sequenceinformation. Nucleic Acids Research, 1996. 24(17): p. 3439-3452.Transcript abundance studies using qPCR also indicated that there is nota Glyma18g02570-like transcript produced by the repeated DNA insertion.Glyma18g02570 transcript abundance was measured using primers (Table 425, 26) that amplify the final two exons and hence should amplify boththe reference genome (full-length; Williams 82-like) Glyma18g02570transcript and possible hybrid Glyma18g02570 transcripts that aretranscribed from DNA that spans the repeat junction. If the repeated DNAproduced an alternative transcript, these primers would amplifyadditional product from genotypes with the repeat. However, nodifferences in transcript abundance were detected between SCN-resistantvs SCN-susceptible varieties using Glyma18g02570 primers 25 and 26.

Protein Structure Prediction and Comparison

The protein structure for the predicted Glyma18g02610 gene product wasmodeled and proteins with the most homologous structures were identifiedusing Phyre2, with default settings. Kelley, L. A. and M. J. E.Sternberg, Protein structure prediction on the Web: a case study usingthe Phyre server. Nature Protocols, 2009. 4(3): p. 363-371.

Methylation Analysis

Locus specific DNA methylation was analyzed using the methylationspecific endonulease McrBC, or the methylation sensitive endonucleaseHpaII followed by PCR. McrBC (New England Biolabs, Ipswich, Mass.)digests DNA with methylated cytosines in a sequence-independent mannerwhile unmethylated DNA is not digested. HpaII (New England Biolabs,Ipswich, Mass.) digests DNA at the recognition sequence CCGG, but HpaIIendonuclese activity is blocked by cytosine methylation. Restrictiondigestions were performed using 600-700 ng of DNA and manufacturer'sprotocols. Control reactions were set up by adding the same amount ofDNA to the reaction buffer with no restriction enzyme. Samples with andwithout the restriction enzyme were incubated at 37° C. for 90 minutes,and heat inactivated at 65° C. for 20 minutes. DNA was visualized in a0.8% ethidium bromide stained gel to ensure DNA digestion. Both digestedand control DNA samples were used for subsequent PCR using GoTaq FlexiDNA polymerase (Promega, Madison Wis.). For DNA treated with McrBC, PCRprimers that spanned methylated DNA would not produce the intendedproduct following PCR because the template DNA would be digested byMcrBC. DNA that was not methylated or not treated with the enzymeyielded a product of the expected size. For DNA treated with HpaII, PCRprimers that spanned the DNA sequence CCGG in which either cytosine wasmethylated yielded a PCR product of the expected size. DNA sequence CCGGthat was not methylated was cleaved by HpaII and failed to yield a PCRproduct. DNA incubated in buffer without HpaII yielded expected PCRproducts. See table in Appendix F and figure for primer details andresults.

Western Blot Analysis

Protein size and abundance were measured using Western blot andimmunodetection procedures (Ausubel et al. 1997). Briefly, protein wasextracted from roots of transgenic soybeans by homogenizing frozen roottissue and re-suspending the material in 2× Tricine sample buffer (0.1 MTrisCl/0.3% SDS pH6.8, 24% glycerol, 8% SDS, 0.2M DTT) at 1:1 w/v ratio.An equal volume of each protein sample was separated in a Tris-Tricinepolyacrylamide gel (9.8% separation gel, 3.9% stacking gel) usingelectrophoresis in the Biorad Mini Protean3 cassette (Biorad, HerculesCalif.). The samples are separated at 35 volts for roughly one hour,followed by another hour at 160 volts. The gel was moved to a transfercassette and aqueous transferred to a Protran nitrocellulose membrane(Whatman, Piscataway, N.J.). The transfer was run for an hour at 80volts at room temperature. Following transfer, membranes were stainedfor total protein using 0.1% Ponceau S (Sigma-Aldrich, St. Louis, Mo.)in 5% acetic acid and imaged. Ponceau S was destained in ddH₂0, and themembrane was blocked over night at 4° C. in TBST (20 mM Tris pH7.5, 8g/L NaCl, 0.1% Tween) carrying 5% milk. New 5% milk in TBST was added tothe membrane and placed on shaker at room temperature for 30 minutes.The membrane was incubated with HA primary antibody directly conjugatedto horse radish peroxidase (HRP) at a 1:1000 concentration in 5% milkTBST for 90 minutes. The membrane was washed 3× in TBST at roomtemperature on a shaker for 20 minutes each. Supersignal West DuraExtended Duration Substrate (Thermo Scientific, Waltham, Mass.) ECL kitwas used following manuacture protocols to detect the HA-HRP antibody onthe membrane. The memebrane was exposed to Cl-Xposure film (ThermoScientific, Waltham, Mass.) and developed.

We claim:
 1. A method of increasing resistance of a plant to nematodescomprising increasing expression of, altering the expression pattern ofor increasing the copy number of a polynucleotide encoding aGlyma18g02580 polypeptide, a Glyma18g02590 polypeptide, or aGlyma18g02610 polypeptide, a polypeptide having 90% or more identity toSEQ ID NO: 1 (2580), SEQ ID NO: 2 (2590), SEQ ID NO: 3 (2610), SEQ IDNO: 5 (2590-88788), SEQ NO: 6 (2590-Peking) or homologs, functionalvariants or combinations of any of the aforementioned polypeptides incells of the plant, wherein increased expression of the polynucleotidecells of the plant increases the resistance of the plant to nematodes.2. The method of claim 1, wherein the expression is increased viagenetic transformation of the plant.
 3. The method of claim 1, whereinthe plant is selected from soybean, sugar beets, potatoes, corn, peasand beans.
 4. The method of claim 1, wherein expression is increased incells in the root of the plant.
 5. The method of claim 1, whereinexpression of the polypeptide is increased by increasing expression ofthe native polynucleotide.
 6. The method of claim 1, wherein expressionof the polypeptide is increased by introducing a construct comprising apromoter operably linked to a polynucleotide encoding the polypeptideinto cells of the plant.
 7. The method of claim 1, wherein expression ofthe polypeptide is increased by incorporation of a transgene comprisinga promoter operably linked to a polynucleotide encoding the polypeptidein the plant.
 8. The method of claim 1, wherein expression of thepolypeptide is increased by introducing one or more copies of apolynucleotide encoding at least two of a Glyma18g02580 polypeptide,Glyma18g02590 polypeptide and a Glyma18g02610 polypeptide.
 9. The methodof claim 1, wherein at least three copies of the polynucleotide areintroduced in the plant.
 10. The method of claim 1, wherein at least tencopies of the polynucleotide are introduced in the plant.
 11. The methodof claim 1, wherein the expression of a Glyma18g02580 polypeptide, aGlyma18g02590 polypeptide and a Glyma18g02610 polypeptide are increased.12. The method of claim 1, wherein increased resistance is measured bythe plant having a lower percentage of invading nematodes that developpast the J2 stage, a lower rate of cyst formation on the roots, reducednematode female index of a plant exposed to nematodes, reduced SCN eggproduction within cysts, reduced overall SCN egg production per plant,or greater yield of soybean seeds on a per-plant basis or aper-growing-area basis as compared to a control plant grown in a similargrowth environment.
 13. A construct comprising a promoter operablylinked to a polynucleotide encoding a Glyma18g02580 polypeptidecomprising SEQ ID NO: 1, a Glyma18g02590 polypeptide comprising SEQ IDNO: 2, 5 or 6, a Glyma18g02610 polypeptide comprising SEQ ID NO: 3 or apolypeptide having at least 90% identity to SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, or a homolog or a functionalportion of any of the aforementioned polypeptides or combinationsthereof.
 14. The construct of claim 13, wherein the promoter is a plantpromoter.
 15. The construct of claim 13, wherein the polynucleotideencodes all three of the polypeptides.
 16. A transgenic cell comprisinga polynucleotide encoding a polypeptide capable of increasing resistanceto nematodes, the polypeptide having at least 90% identity to aGlyma18g02580 polypeptide comprising SEQ ID NO: 1, a Glyma18g02590polypeptide comprising SEQ ID NO: 2, 5 or 6, a Glyma18g02610 polypeptidecomprising SEQ ID NO: 3 or a polypeptide having at least 90% identity toSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, ora homolog or functional portion thereof or combinations thereof.
 17. Thetransgenic cell of claim 16, wherein the polynucleotide encodes at leasta Glyma18g02610 polypeptide, a Glyma18g02590 polypeptide and a.Glyma18g02580 polypeptide.
 18. The transgenic cell of claim 16, whereinthe polynucleotide is present in at least three copies in the plant. 19.The transgenic cell of claim 16, wherein the polynucleotide is presentin at least ten copies in the plant.
 20. A seed comprising thetransgenic cell of claim
 16. 21. A plant grown from the seed of claim20.
 22. A transgenic plant comprising the cell of claim
 16. 23. A part,progeny or asexual propagate of the transgenic plant of claim
 22. 24. Amethod of generating a transgenic plant with increased resistance tonematodes comprising introducing an exogenous polynucleotide encoding aGlyma18g02580 polypeptide having at least 90% identity to SEQ ID NO: 1,Glyma18g02590 polypeptide having at least 90% identity to SEQ ID NO: 2,5 or 6, or Glyma18g02610 polypeptide having at least 90% identity to SEQID NO: 3, or a homolog, functional variant or combinations thereof intoa soybean plant cell or progeny thereof, whereby expression of thepolypeptide is increased in a root cell of the soybean plant and wherebythe plant has increased resistance to nematodes as compared to a controlplant.
 25. The method of claim 24, wherein the polynucleotide encodes atleast a Glyma18g02610 polypeptide, a Glyma18g02590 polypeptide and aGlyma18g02580 polypeptide.
 26. The method of claim 24, wherein thepolynucleotide is present in at least three copies in the plant.
 27. Amethod of screening a first plant cell for resistance or susceptibilityto nematodes comprising: detecting a genetic marker or selectable markerin the first plant cell associated with cyst nematode resistance orsusceptibility to cyst nematodes; and predicting the resistance orsusceptibility of the first plant cell to nematodes, wherein the geneticmarker is selected from methylation status of the promoter, upstreamregion or gene body of at least one of Glyma18g02610, Glyma18g02590, orGlyma18g02580; the level of RNA transcript, transcription or proteinexpression of at east one of Glyma18g02610, Glyma18g02590, Glyma18g02580or Glyma18g02600; the genomic copy number of at least one ofGlyma18g02610, Glyma18g02590, Glyma18g02580, Glyma18g02600 or anyportion of the 31.2 kb Rhg1 region identified as being present inmultiple copies in resistant varieties; the genomic DNA segment carryinga repeat junction between Glyma18g02610 and Glyma18g02570; thetranscription or expression level of at least one of Glyma18g02610,Glyma18g02590, Glyma18g02600, or Glyma18g02580 after or upon contactwith a nematode; a single nucleotide polymorphism in at least one ofGlyma18g02610, Glyma18g02590, Glyma18g02580, Glyma18g02600 or within the31.2 kb Rhg1 repeat region; the presence or absence of more than onecopy of a genomic region comprising at least one of Glyma18g02600,Glyma18g02610, Glyma18g02590, or Glyma18g02580; and wherein thepredicting step comprises comparing the marker in the first plant cellto the marker in a second plant cell with a known resistance orsusceptibility phenotype, wherein the phenotype of the second cell ispredictive of the phenotype in the first cell.
 28. The method of claim27, wherein detecting comprises amplifying the marker or a portion ofthe marker to produce an amplified product and determining all or partof the DNA sequence of the amplified product or assessing the markerusing differential sensitivity to a restriction endonuclease, allelespecific hybridization analysis, allele specific PCR, high densitynucleotide array analysis, quantitative PCR, Northern blot analysis,microsatellite analysis, ELISA or Western blot analysis.
 29. The methodof claim 27, wherein the prediction is used to select resistant plantcells for use in developing resistant soybean lines.
 30. A method ofincreasing resistance of a plant to nematodes comprising expressing apolynucleotide encoding a Glyma18g02580 polypeptide of SEQ ID NO: 1, aGlyma18g02590 polypeptide of SEQ ID NO: 2, 5 or 6, or a Glyma18g02610polypeptide of SEQ ID NO: 6, a polypeptide having 90% or more identityto SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO:6, or homolog, a functional variant or combination of any of theaforementioned polypeptides in a cell, and applying the polypeptide or acell comprising the polypeptide to the plant, seeds of the plant or soilin which the seeds may be planted, wherein the application increases theresistance of the plant to nematodes.
 31. The method of claim 30,wherein the polynucleotide encodes a Glyma18g02610 polypeptide,Glyma18g02590 polypeptide, and a Glyma18g02580 polypeptide.